JP2017505606A - Pseudotype lentiviral vector - Google Patents

Abstract

The invention relates to a nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding the vesicular stomatitis virus envelope glycoprotein (VSV-G) linked to a (poly) peptide comprising or consisting of a cell membrane binding domain, The nucleic acid sequence in the 5 ′ to 3 ′ direction, (a) a first sequence segment encoding an endoplasmic reticulum (ER) signal sequence; (b) comprising or consisting of a cell membrane binding domain A nucleic acid molecule comprising: a) a second sequence segment encoding a peptide; (c) a third sequence segment encoding a linker; and (d) a fourth sequence segment encoding said VSV-G. Furthermore, the present invention provides a vector comprising the nucleic acid molecule of the present invention, a host cell comprising the vector or the nucleic acid molecule, a polypeptide encoded by the nucleic acid molecule, and a method for producing a polypeptide encoded by the nucleic acid molecule. About. In addition, the present invention relates to pseudotyped lentiviral vector particles, methods for transducing cells, and kits comprising various combinations of the nucleic acid molecules, vectors, polypeptides and host cells of the present invention.

Description

  The invention relates to a nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding the vesicular stomatitis virus envelope glycoprotein (VSV-G) linked to a (poly) peptide comprising or consisting of a cell membrane binding domain, Wherein said nucleic acid sequence comprises, in 5 ′ to 3 ′ direction, (a) a first sequence segment encoding an endoplasmic reticulum (ER) signal sequence; (b) comprising or consisting of a cell membrane binding domain A nucleic acid molecule comprising: a second sequence segment encoding a (poly) peptide of: (c) a third sequence segment encoding a linker; and (d) a fourth sequence segment encoding said VSV-G. . Furthermore, the present invention provides a vector comprising the nucleic acid molecule of the present invention, a host cell comprising the vector or the nucleic acid molecule, a polypeptide encoded by the nucleic acid molecule, and a method for producing a polypeptide encoded by the nucleic acid molecule. About. In addition, the present invention relates to pseudotyped lentiviral vector particles, methods for transducing cells, and kits comprising various combinations of the nucleic acid molecules, vectors, polypeptides and host cells of the present invention.

  In this specification, a number of documents including patent applications and manufacturer's manuals are cited. The disclosures of these documents are not considered relevant to the patentability of the present invention, but are hereby incorporated by reference in their entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

  Lentiviral expression vectors provide stable gene expression and have become an important tool for research and gene therapy applications. They make it possible to integrate the gene of interest into the genome of a wide range of mitotic cells and non-dividing target cells [1]. Gamma retroviral vectors and lentiviral vectors have been successfully used in several gene therapy clinical trials. With the recent success reported for gene therapy of severe combined immunodeficiency symptoms (SCID) and tumor therapy for transcription of chimeric T cell receptors [2-5], a third safety profile has been enhanced. The number of clinical applications of generational self-inactivating lentiviruses is expected to increase.

  Lentiviral vectors are often pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) that binds to the ubiquitously expressed LDL receptor [6]. VSV-G pseudotyped lentiviral vectors have excellent mechanical stability, which allows spinoculation and production of high titer vector stock [7]. The VSV-G protein is directed to the endoplasmic reticulum by a signal sequence (SS) and is glycosylated in the endoplasmic reticulum to produce a trimer, which is then incorporated into the cell membrane. Changes in the protein structure of VSV-G generally lead to inappropriate processing and unstable lentiviruses [8]. Despite advances in cell transduction methods, low transduction rates have been reported in several cell types, such as lymphoid cells, including primary T cells and lymphoma cells, and several epithelial cell lines [4, 9]. This low transduction rate has the disadvantage that it necessitates the use of a high multiplicity of infection (MOI). In order to increase the contact time and lentiviral uptake rate, genetic modification of the lentiviral VSV-G envelope for specific antigen binding has been reported [10-13]. An intact antibody or antibody fragment can function as a high affinity connector between the viral and cell membranes, thereby increasing transduction rates. However, previous attempts to fuse scFv N-terminal fusions to VSV-G resulted in defective lentiviral vectors that lost their transduction activity as described in [14]. Thus, another approach was developed [12] aimed at modifying the surface of lentiviral vectors with a smaller antibody-binding ZZ domain from staphylococcal protein A fused to VSV-G. Although this approach has been shown to be successful in vitro, two reagents, the modified lentiviral vector and each antibody, need to be approved, provided in clinical grade, and combined to produce an active therapeutic agent. Therefore, it is not very advantageous as a clinical protocol development.

Thus, although there is the fact that much effort has been expended in methods of establishing alternative and / or ameliorative means and methods of cell transduction, there remains a need to provide such methods.
This need is addressed by providing the aspects characterized in the claims.

  Accordingly, the present invention is a nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding the vesicular stomatitis virus envelope glycoprotein (VSV-G) linked to a (poly) peptide comprising or consisting of a cell membrane binding domain. Wherein said nucleic acid sequence comprises, in the 5 'to 3' direction, (a) a first sequence segment encoding an endoplasmic reticulum (ER) signal sequence; (b) comprising or consisting of a cell membrane binding domain (C) a third sequence segment encoding a linker; (d) a third sequence segment encoding a linker; and (d) a fourth sequence segment encoding said VSV-G.

  The articles “a” and “an” are used herein to refer to one or more (ie, at least one) of the grammatical objects of the article.

According to the present invention, nucleic acid molecules, also referred to herein as polynucleotides or nucleic acid sequences, include DNA and RNA, such as, for example, cDNA or genomic DNA. As used herein, the term “RNA” is understood to include all forms of RNA, including genomic RNA, such as in the case of mRNA, tRNA and rRNA, and RNA of RNA viruses. Embodiments describing “RNA” preferably include mRNA. Also included are nucleic acid mimetic molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA, mixed polymers, both sense and antisense strands. As will be readily appreciated by those skilled in the art, nucleic acid molecules can include additional non-natural or derivatized nucleotide bases. Such nucleic acid mimic molecules or nucleic acid derivatives in the present invention include peptide nucleic acid (PNA), phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2′-O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), locked Nucleic acids (LNA) and RNA derivatives in which the ribose ring is constrained by a methylene bond between the 2′-oxygen and the 4′-carbon (see, eg, Braasch and Corey, Chemistry & Biology, 8, 1-7 (2001 )) Is included. PNA is an amide in place of the sugar-phosphate backbone of DNA or RNA as described by Nielsen et al, Science, 254: 1497 (1991); and Egholm et al, Nature 365: 666 (1993). A synthetic DNA mimic with a backbone.
In a preferred embodiment, at least the nucleic acid sequences specifically described in options (a) to (d) are DNA. In an even more preferred embodiment, all nucleic acid molecules of the present invention are DNA.

  The nucleic acid molecules of the present invention may be of natural origin or of synthetic or semi-synthetic origin. Thus, the nucleic acid molecule may be, for example, a nucleic acid molecule synthesized according to the usual protocol of organic chemistry. Those skilled in the art are familiar with the preparation and use of such nucleic acid molecules (see, eg, Sambrook and Russel "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001)).

  As used herein, the term “comprising” means that additional sequences, components and / or method steps can be included in addition to the specifically described sequences, components and / or method steps. However, this term also encompasses that the claimed subject matter consists of just the sequences, components and / or method steps described.

In embodiments in which the nucleic acid molecule comprises (rather than consists of) the described sequence, additional nucleotides are extended on that particular sequence, either at the 5 'end or at the 3' end, or both. These additional nucleotides may be heterologous or homologous. In the case of homologous sequences, these sequences may contain no more than 1500 nucleotides at the 5 ′ end and / or the 3 ′ end, for example no more than 1000 nucleotides at the 5 ′ end and / or the 3 ′ end. E.g. 900 or fewer nucleotides, more preferably 800 or fewer nucleotides, e.g. 700 or fewer nucleotides, e.g. 600 or fewer nucleotides, e.g. 500 or fewer nucleotides, even more preferably 400 The following nucleotides, for example 300 or fewer nucleotides, for example 200 or fewer nucleotides, for example 100 or fewer nucleotides, more preferably 50 or fewer nucleotides, for example 40 or fewer nucleotides, for example 30 The following nucleotides, for example 20 nucleotides or less Preferably it may contain no more than 10 nucleotides, most preferably no more than 5 nucleotides. As used herein, the term “... Or less nucleotides” refers to the number of nucleotides including any integer less than or equal to the specifically stated number. For example, the term “5 or fewer nucleotides” refers to any of 1, 2, 3, 4, and 5 nucleotides. Furthermore, in the case of homologous sequences, these embodiments do not include whole genomes or whole chromosomes.
Additional heterologous sequences include, for example, heterologous promoters operably linked to the coding sequences of the invention, and regulatory nucleic acid sequences that are well known in the art and described in more detail herein. May be included.

  The nucleic acid sequence contained in or constituting the nucleic acid molecule of the present invention encodes the vesicular stomatitis virus envelope glycoprotein linked to a (poly) peptide comprising or consisting of a cell membrane binding domain. Thus, the nucleic acid sequence, and thus the nucleic acid molecule of the invention, encodes a fusion protein. In the present specification, this fusion protein is also referred to as “the fusion protein of the present invention”.

  According to the present invention, the nucleic acid molecule is composed of at least four sequences defined by options (a) to (d), wherein the order of these four nucleic acid sequences is indicated from 5 ′ to 3 ′. That is, the first nucleic acid sequence is the sequence of (a), followed by (b), followed by (c), and similarly (d).

  The first sequence described in option (a) is a nucleic acid sequence encoding an endoplasmic reticulum (ER) signal sequence. The ER signal sequence is a short peptide sequence present at the N-terminus of a newly synthesized protein. At the start of mRNA translation, this sequence is the first sequence to be translated and the new signal sequence of the nascent protein binds to signal recognition particles (SRPs). When SRPs bind to the ER signal sequence, translation is temporarily stopped, the SRP-signal sequence-mRNA-ribosome complex is transferred to the ER, and the SRP recognizes a receptor (RER) on the ER membrane. ,Join. Subsequently, the signal sequence is inserted into the RER and traverses the membrane, resulting in continued translation while a new polypeptide chain is drawn into the ER lumen.

Sequence motifs for the ER signal sequence are well known in the art and are described, for example, in Lemberg and Martoglio, or Schwartz [45, 46]. Thus, one skilled in the art knows the appropriate naturally occurring ER signal sequence or is in a position to generate an artificial or non-naturally occurring ER signal sequence that exhibits the aforementioned functions and can be used in the present invention. is there.
In order to produce the fusion protein encoded by the nucleic acid molecule of the present invention, preferably a naturally occurring ER signal sequence is used, more preferably a viral ER signal sequence or an ER signal sequence endogenous to the cell line is used. . In the case of a viral ER signal sequence, it is preferably derived from Rhabdoviridae, more preferably from the genus Becyclovirus. Most preferably, the ER signal sequence is an ER signal sequence that endogenously mediates the transfer of VSV-G used in option (d) to the ER.

  The second sequence segment described in option (b) is a nucleic acid sequence encoding a (poly) peptide comprising or consisting of a cell membrane binding domain.

  The term “(poly) peptide” according to the invention relates to polypeptides and peptides. The term “polypeptide” used interchangeably with the term “protein” herein refers to a linear molecular chain of amino acids, including a single chain protein or a fragment thereof containing more than 30 amino acids, The term “peptide” as used herein refers to a group of molecules consisting of up to 30 amino acids. The (poly) peptide may further form an oligomer composed of at least two identical or different molecules. Corresponding higher order structures of such multimers are accordingly referred to as homodimers, heterodimers, homotrimers or heterotrimers. Such multimers are also included in the definition of the term “(poly) peptide”. The terms “polypeptide” and “peptide” also refer to naturally modified polypeptides / peptides that are achieved, for example, by glycosylation, acetylation, phosphorylation, and similar modifications well known in the art. .

  According to the invention, a nucleic acid molecule encodes a “(poly) peptide comprising or consisting of a cell membrane binding domain”. The term “cell membrane” and its scientific meaning related to structure and function are well known in the art [47, 48] and are used accordingly in the context of the present invention. The term “cell membrane binding domain” in the present invention may be any amino acid sequence capable of directly binding to the cell membrane. Binding domains that bind only indirectly to the cell membrane via an intermediate molecule are specifically excluded.

  The function of the cell membrane binding domain within the fusion protein of the present invention is to act as a connector between the viral particle expressing the fusion protein of the present invention and the target cell transduced by the viral particle. Therefore, it is understood that a cell membrane binding domain that can bind to the cell membrane of the target cell of interest should be selected. Preferably, the cell membrane binding domain is capable of binding to a mammalian target cell membrane. More preferably, the cell membrane binding domain binds to the cell membrane of a human target cell such as, for example, a progenitor cell, disease cell, primary cell line, epithelial cell, endothelial cell, neuronal cell, lymphoid cell, stem cell or tumor cell. it can.

The term “binding” in the context of the present specification refers to the ability of a domain to bind to the cell membrane, for example via covalent or non-covalent interactions. A “covalent” interaction is a form of chemical bonding characterized by the sharing of electron pairs between atoms or between atoms and other covalent bonds. Covalent bonds include many types of interactions well known in the art such as σ bonds, π bonds, metal-nonmetal bonds, agostic interactions, and three-center two-electron bonds. A “non-covalent” bond is a chemical bond that does not involve the sharing of an electron pair. Non-covalent bonds are important in maintaining the three-dimensional structure of macromolecules such as proteins and nucleic acids, and are involved in many biological processes in which molecules bind specifically to each other, but transiently. Yes. There are several types of non-covalent bonds such as hydrogen bonds, ionic interactions, van der Waals interactions, charge-charge bonds, charge-dipole bonds, dipole-dipole bonds and hydrophobic bonds. Non-covalent interactions often include several different types of cooperating non-covalent bonds, eg, to maintain the ligand at the location of the target binding site on the cell membrane. Interactions can occur involving groups such as charges or dipoles that can occur many times on the surface of the cell membrane.
Preferably, the interaction (ie binding) occurs at a defined site in the cell membrane (including a specific cell membrane component / epitope) and the formation of at least one interaction, preferably a network of several specific interactions Proceed with formation. Even more preferably, the binding is specific for the target cell. That is, binding occurs at the cell membrane of the target cell and does not occur or does not occur significantly at the cell membrane of the non-target cell.

  The structure of the domain is not limited as long as the domain has a function of binding to the cell membrane, preferably a function of specific binding. A variety of cell membrane binding domains are known in the art, such as antibodies and fragments thereof, protein scaffolds, and cell binding such as (poly) peptides [49] from fibronectin and (poly) peptides having heparin binding activity. Protein domains with properties are included, but are not limited to these.

The term “antibody” as used in accordance with the present invention includes polyclonal antibodies, monoclonal antibodies, and derivatives or fragments thereof that still retain their binding specificity. Antibody fragments or derivatives include, inter alia, single domain antibodies, Nanobodies, camel V _H H fragments, and VNAR fragments derived from cartilaginous Fab or Fab ′ fragments, and Fd, F (ab ′) ₂ , Fv or scFv fragments. (See, eg, Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1988; Harlow and Lane “Using Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1999). The term “antibody” also includes embodiments such as chimeric antibodies (human constant domains, non-human variable domains), single chain antibodies, and humanized antibodies (human antibodies with the exception of non-human CDRs). Preferably, the antibody is a humanized antibody.

  Various techniques for the production of antibodies are well known in the art, eg, Altshuler et al., 2010 (Altshuler EP, Serebryanaya DV, Katrukha AG, 2010, Biochemistry (Mosc), Vol. 75 (13), 1584. )It is described in. Polyclonal antibodies can thus be obtained from the blood of animals after immunization with antigen mixed with additives and adjuvants, and monoclonal antibodies can be produced by any technique that provides antibodies produced by continuous cell line culture. can do. Examples of this technique are described, for example, in Harlow E and Lane D, Cold Spring Harbor Laboratory Press, 1988; Harlow E and Lane D, “Using Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press, 1999, Hybridoma technology, trioma technology, human B cell hybridoma technology described in Kohler and Milstein, 1975 (see, for example, Kozbor D, 1983, Immunology Today, vol.4, 7; Li J, Sai T, Berger M, Chao Q , Davidson D, Deshmukh G, Drozdowski B, Ebel W, Harley S, Henry M, Jacob S, Kline B, Lazo E, Rotella F, Routhier E, Rudolph K, Sage J, Simon P, Yao J, Zhou Y, Kavuru M, Bonfield T, Thomassen MJ, Sass PM, Nicolaides NC, Grasso L., 2006, PNAS, vol. 103 (10), 3557), and EBV-hybridoma technology for producing human monoclonal antibodies (Cole et al, 1985, Alan R. Liss, Inc, 77-96). Furthermore, recombinant antibodies can be obtained from monoclonal antibodies, or can be newly prepared using various display methods such as phage display, ribosome display, mRNA display, or cell display. Suitable systems for the expression of recombinant (humanized) antibodies can be selected from, for example, bacteria, yeast, insects, mammalian cell lines, or transgenic animals or plants (see, eg, US patents). 6,080,560; Holliger P, Hudson PJ. 2005, Nat Biotechnol., Vol. 23 (9), 11265). Furthermore, techniques described for the production of single chain antibodies (see, in particular, US Pat. No. 4,946,778) can be adapted to produce single chain antibodies specific for the targets of the present invention. By using the surface plasmon resonance used in the BIAcore system, the efficiency of the phage antibody can be increased. Preferably, the antibody is selected from the group consisting of anti-EGFR antibody, anti-CD30 antibody, anti-CD34 antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD19 antibody, anti-CD20 antibody or anti-CD44 antibody. More preferably, the antibody is an anti-EGFR antibody or an anti-CD30 antibody. It is known that epidermal growth factor receptor (EGFR) is mainly expressed on the epithelium and CD30 is expressed on lymphoma cells [15-18]. A variety of antibody fragments with high affinity for EGFR and CD30 are available and have been used previously in vitro and in vivo [19-21]. Preferably, the at least one affinity reagent may be one of the anti-EGFR or anti-CD30 antibodies used in the examples. Suitable scFv antibodies against CD30 and EGFR are known in the art and are described, for example, in Klimka et al. [63] and Kettleborough et al. [64].

  The term “protein scaffold” is known in the art and is a new type of derivative derived from a small, robust non-immunoglobulin “scaffold” that could be provided with a given binding function using combinatorial protein design methods. It relates to a receptor protein (Gebauer and Skerra (2009) Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol, 13, 245-255). Non-limiting preferred examples of engineered protein scaffolds are protein scaffolds for the development of drug candidates for therapy or in vivo diagnosis, and are described in further detail below: Adnectin, Affibody, Anticalin, DARPin and designed Kunitz-type inhibitors are included.

  The nucleic acid molecule of option (b) can encode a (poly) peptide comprising said cell membrane binding domain or a (poly) peptide consisting of said cell membrane binding domain. In the first option, the cell membrane binding domain is part of a larger molecule such as an antibody. In the latter option, the (poly) peptide is itself a cell membrane binding domain. Examples of this option include, but are not limited to, the protein scaffolds described herein above. In either case, the nucleic acid molecule of option (b) is encoded by the nucleic acid molecule of the present invention, regardless of whether the cell membrane binding domain is part of a larger molecule or the cell membrane binding domain itself. It is understood that it comprises a part of the protein, ie at least the additional segments (a), (c) and (d).

  The third sequence segment in option (c) encodes a “linker”. The term “linker” as used in the present invention relates to subsequent amino acids (ie, peptide linkers). In accordance with the present invention, the linker is located between the amino acid sequence encoded by the second sequence segment and the amino acid sequence encoded by the fourth sequence segment, thereby linking these two sequences in the translated protein. Connect (link).

  The linker envisaged by the present invention is a (poly) peptide linker of at least 3 amino acids in length. In a preferred embodiment, the linker length is 3-100 amino acids, more preferably the linker length is 5-50 amino acids, even more preferably the linker length is 10-20 amino acids. is there. Most preferably, the linker length is 13 amino acids. In an alternative or additional embodiment, the linker provides a distance of the cell membrane binding domain from the surface of the lentiviral vector to about 5-100 mm, more preferably about 5-75 mm, and even more preferably about 5-50 mm. Extend to. Most preferably, the linker extends the distance of the cell membrane binding domain to about 30 mm. Means and methods for determining the distance of the cell membrane binding domain from the surface of the lentiviral vector are known in the art and include three-dimensional structure of the fusion protein, prediction of secondary / tertiary structure, homology modeling, and This includes, but is not limited to, analysis of sequence alignments.

  It will be appreciated that the linker is useful for physically separated nucleic acid segments, such as segments (b) and (d) of the nucleic acid molecules of the present invention. As is known in the art, the nature of the linker, ie length and / or amino acid sequence, can affect the expressed fusion protein. For example, the nature of the linker can alter and enhance the stability and / or solubility of the expressed fusion protein or have a steric effect that affects the interaction or multimerization with other molecules. Yes. Thus, since it is a requirement that the polypeptides of the invention maintain the ability to bind to target cells, preferred linkers are the correct expression and folding of the encoded polypeptide, and the organism of the VSV-G and cell membrane binding domain. Must not interfere with the scientific function. Thus, the linker must be long enough and / or flexible to allow these properties as before. Thus, the length and sequence of the linker can be varied based on the actual composition, ie the choice of the sequence segment of the molecule of the invention. Furthermore, preferred linkers must adopt a flexible structure and should minimize hydrophobic or charged properties to avoid interaction with functional protein domains and / or solvents. Preferably, the linker is selected to be a moiety that can avoid detection by the immune system. Those skilled in the art know how to design appropriate linker molecules based on their general knowledge. For example, peptide linkers can be selected from the commercially available LIP (Loop in Proteins) database (Michalsky et al., 2003 [50]) (see, eg, Glen Research (22825 Davis Drive , Sterling, Virginia, 20164 USA).

  Those skilled in the art are well aware of how to test the compatibility of different linkers. For example, linker compatibility is easily tested by comparing the virus yield and / or transduction rate of the lentiviral vectors of the present invention as described herein (see Example 4). Can do.

  Preferably, the linker is a flexible linker comprising, for example, the amino acids glycine, asparagine and / or serine, and preferably the linker comprises the amino acids glycine and serine or alanine and serine. More preferably, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO: 3. Even more preferably, the linker consists of the amino acid sequence shown in SEQ ID NO: 3.

  The fourth sequence segment in option (d) encodes the vesicular stomatitis virus envelope glycoprotein. Vesicular stomatitis virus (abbreviated herein as VSV), also referred to as vesicular stomatitis Indianavirus (VSIV), is a virus of the group V rhabdoviridae (ie, negative sense single-stranded RNA). Viruses, their hosts, and diseases induced by infection with the viruses are well known in the art [51-53]. VSV-G is a transmembrane glycoprotein [6,52] and is immature and non-infectious constructed by packaging cells that produce infectious pseudotyped virus in cell-free conditions in vitro. It is known to bind efficiently to envelope-deficient retrovirus-like particles. Thus, the sequence of the VSV envelope glycoprotein is well known in the art and, according to the present invention, the cytoplasmic tail, the transmembrane domain, and the membrane proximal extracellular stem region that mediates efficient viral budding. (External domain or extracellular domain). The sequence of VSV-G has been described in the art, eg [54], and the natural VSV-G NCBI reference number is NP_955548.1 (NCBI Genome Project, 2000).

  It is understood that the first sequence segment in the present invention already encodes the ER signal sequence. Thus, the VSV-G encoded by the fourth sequence segment preferably does not contain an additional ER signal sequence. For the purposes of the present invention, either the native VSV-G sequence or the modified VSV-G sequence [55, 56] can be used. For example, introduction of a target peptide such as RGD or a positively charged peptide (for example, polylysine peptide (K7 to K20)) into the permissible epitope insertion site of VSV-G (see, [55 and 56]). Can be used to achieve improved effects [57-60]. When using a modified VSV-G sequence, the ability of the modified VSV-G to bind to the LDL receptor and / or a stable and correctly prepared wrench pseudotyped with said modified VSV-G sequence It is important not to impair the ability to provide viral vectors. Preferably, the modification is a transmembrane domain (eg, a range from position 426 to 466 of the VSV-G protein represented by SEQ ID NO: 6) or a cytoplasmic domain (eg, position 467 of the VSV-G protein represented by SEQ ID NO: 6). (In the range of ~ 495). Further, the modification is preferably not in the region between amino acids 385 and 444 of the VSV-G glycoprotein represented by SEQ ID NO: 6. This is because this region has a tendency to form an α-helix, suggesting that this region may allow direct interaction with the membrane [62]. Preferably, the sequence of VSV-G is not altered. In addition, it is particularly preferred that the N-terminal domain of VSV-G is directly attached to the linker as described herein above.

In accordance with the present invention, the sequence segments are arranged in the 5 ′ to 3 ′ direction. Each sequence segment can be continued to the previous sequence segment with or without an intermediate sequence. Terminal sequences can be added alternatively or additionally to the 5 ′ end and / or the 3 ′ end, as described herein above. For example, it is clearly understood herein that a fifth sequence segment encoding a detectable label (ie, the 3 ′ end of the fourth segment) is included in the nucleic acid molecules of the invention. Exemplary detectable labels, and exemplary intermittent sequences can be, for example, tags for subsequent purification or detection of any of the nucleic acid molecules of the invention or polypeptides encoded thereby. . Non-limiting examples of tags include strep-tags, chitin-binding protein (CBP), maltose-binding protein (MBP), glutathione-S-transferase (GST), FLAG tag, HA tag, Myc tag, poly (His) tags, and derivatives thereof, or epitope tags such as, for example, V5 tag, C-myc tag and HA tag are included. All these tags and their derivatives are well known in the art and are described, for example, in Lichty JJ et al. Comparison of affinity tags for protein purification Protein Expr Purif. 2005 May; 41 (1): 98-105. Yes. Preferably, when the tag is included in the fusion protein, the tag is the His tag shown in FIG. Detectable labels further include, but are not limited to, radioactive labels such as ³ H or ³² P, fluorescent labels, and reporter proteins, for example. Nucleic acid labeling is well understood in the art and is described, for example, in Sambrook and Russel "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, NY (2001).

Where intermediate sequences and / or terminal sequences are present, these increase the integrity and functionality of the VSV-G fusion proteins of the invention, particularly in transfection efficiency into cells, preferably cells that are difficult to transfect. It is chosen so as not to impair its use as part of the leading lentiviral vector. Based on its scientific knowledge, those skilled in the art are aware of modifications that may adversely affect the integrity and functionality of the fusion protein containing the cell membrane binding domain, thereby allowing appropriate intermittent sequences as needed. And / or terminal sequences can be identified. In addition, one skilled in the art will ascertain experimentally, for example, based on the experiments described herein, whether the integrity and functionality are impaired by the addition of intermittent and / or terminal sequences. Can do.
Preferably, no intermediate sequence is present between the second, third and fourth sequence segments. That is, these sequence segments follow each other directly such that the fusion proteins corresponding to the second, third and fourth sequence segments are composed only of nucleotides belonging to the second to fourth sequence segments.

  In accordance with the present invention, a novel VSV-G fusion protein comprises an N-terminal single chain antibody fragment (scFv) against either the tumor antigen EGFR or CD30, wherein VSV-G and scFv are separated by a flexible linker Is done. As shown in the appended examples, high titer lentiviral vector (LV) preparations are obtained using wt-VSV-G and VSV-G modified with scFv of the present invention at a specific ratio I was able to. In addition, as shown in the appended examples, when lentiviral particles having no VSV-G fusion protein without a linker were produced, the virus yield was strongly reduced. The introduction rate could not be increased. Therefore, the inventors conclude that the linker sequence between the cell membrane binding domain and VSV-G is essential for the function of lentiviral particles retargeted with the VSV-G fusion protein.

  To date, no successful attempt to attach the scFv N-terminus to VSV-G has been reported in the art. Instead, an alternative approach performed in the art is to modify the surface of a lentiviral vector with a small antibody-binding ZZ domain, eg, derived from staphylococcal protein A fused to VSV-G [12]. . However, this approach requires that the modified lentiviral vector and each antibody's two reagents be approved for clinical use, provided in clinical grade purity, and combined to produce an active therapeutic agent. Therefore, the novel VSV-G fusion protein of the present invention has an improved transduction rate at a low MOI, and is suitable for transduction of cells that are difficult to transduce, such as lymphoid cells or tumor cells. Provides an improved means for pseudotyping LV to prepare lentiviral particles.

In a preferred embodiment of the nucleic acid molecule of the present invention, the (poly) peptide comprising or consisting of the cell membrane-binding domain encoded by the second sequence segment is a single chain antibody, a single domain antibody, a V _H H antibody Selected from the group consisting of a fragment, a VNAR single chain antibody and a protein scaffold.

  As described herein above, the types of antibodies include embodiments such as chimeric antibodies (human constant domains, non-human variable domains) and humanized antibodies (human antibodies with the exception of non-human CDRs). .

  The term "protein scaffold" used interchangeably with the term "designed protein scaffold" herein is well known in the art and has a defined binding function using combinatorial protein design methods; A new generation of receptor protein derived from a small and robust non-immunoglobulin “scaffold” (Gebauer and Skerra (2009) Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol, 13, 245-255). Examples of preferred and non-limiting designed protein scaffolds include Adnectin, Affibody, Anticalin and DARPin.

  In the present invention, “Adnectin” (also referred to as a monobody) is a human having a 94-residue Ig-like β-sandwich folded structure with 2-3 exposed loops and lacking a central disulfide bridge. Based on the 10th extracellular domain of fibronectin III (10Fn3).

  In the present invention, an “affibody” is based on the Z domain of staphylococcal protein A, ie, about 58 residues that bind together three helices and provide an interface on two α-helices.

  The term “anticarin” as used herein refers to a designed protein derived from lipocalin (Beste, G., Schmidt, FS, Stibora, T., Skerra, A. (1999) Small antibody-like proteins. with prescribed ligand specificities derived from the lipocalin fold. PNAS, 96, 1898-903). Anticalin forms a highly conserved core in lipocalin and uses an eight-strand β-barrel that naturally forms a binding site for the ligand using four structurally variable loops at the open end. Have Anticarins are not homologous to the IgG superfamily, but have previously been considered common for antibody binding sites: (i) high structural flexibility as a result of sequence variation, and (ii) Shows increased conformational flexibility to guide the fit to different shaped targets.

  In the present invention, the term “DARPin” refers to an ankyrin repeat domain (166 residues) designed to provide a rigid interface typically resulting from three repeated β-turns. DARPin typically carries 3 iterations corresponding to an artificial consensus sequence where 6 positions are randomized per iteration. As a result, DARPin loses structural flexibility.

  A “Kunitz domain peptide” is derived from the Kunitz domain of a Kunitz-type protease inhibitor, such as, for example, bovine pancreatic trypsin inhibitor (BPTI), amyloid precursor protein (APP) or tissue factor pathway inhibitor (TFPI). The Kunitz domain has a molecular weight of about 6 kDa, and a domain having the necessary target specificity can be selected by a display technique such as phage display (Gebauer and Skerra (2009) Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol, 13, 245-255).

In a particularly preferred embodiment of the nucleic acid molecule of the invention, the (poly) peptide comprising or consisting of a cell membrane binding domain encoded by the second sequence segment is a single chain antibody.
For the arrangement of the V _L domain and V _H domain of an immunoglobulin domain, V _L domain may be located at the N-terminus or C-terminus of the V _H domain. Accordingly, in the nucleic acid molecule of the present invention, the nucleic acid encoding the _VL domain can be located 5 ′ or 3 ′ of the nucleic acid encoding the _VH domain. One skilled in the art can determine what arrangement of the _VH and _VL domains is more suitable for a specific scFv.

  In a further preferred embodiment of the nucleic acid molecule of the invention, the (poly) peptide comprising or consisting of a cell membrane binding domain is one or more selected from the group consisting of glycolipids, phospholipids, oligosaccharides and proteins. It binds specifically to cell membrane components.

  A glycolipid is a lipid to which carbohydrates present on a cell membrane are bound, and is useful for cell recognition among other components on the cell membrane. Non-limiting examples of glycolipids include glyceroglycolipid, sphingoglycolipid and glycosylphosphatidylinositol.

  Phospholipids are amphiphilic lipids that contain a phosphate group. The most commonly found phospholipids in cell membranes are phosphatidylcholine (lecithin, abbreviated PC), phosphatidylethanolamine (cephalin, abbreviated PE), phosphatidylserine (PS) and sphingomyelin. Based on their chemical composition, these fall into two groups: glycerophospholipids with glycerin as the basic structure and sphingomyelin, a sphingolipid containing a sphingosine-derived phosphate.

  Oligosaccharides are saccharide polymers that contain a small number of simple sugars (monosaccharides). They are generally found on the plasma membrane of animal cells and play a role in cell-cell recognition among other components on the plasma membrane.

  As described herein above, a protein is a linear chain of amino acids containing 30 or more amino acids. Suitable proteins in this embodiment are G protein-coupled cell receptors (GPCRs), clusters of differentiation (CD); also referred to in the art as cluster of designations, cell surface proteins, cell surface receptors or cells It is a surface co-receptor.

  In a further preferred embodiment of the nucleic acid molecule of the present invention, (a) the first sequence segment encoding the ER signal sequence has at least 60% homology with the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 1 Or (b) the third sequence segment encoding the linker comprises the nucleic acid sequence shown in SEQ ID NO: 3 or a nucleic acid sequence having at least 60% homology with SEQ ID NO: 3 or And / or (c) the fourth sequence segment encoding said VSV-G comprises the nucleic acid sequence shown in SEQ ID NO: 5 or a nucleic acid sequence having at least 60% homology with SEQ ID NO: 5 or Consists of.

Accordingly, included in the present invention are nucleic acid molecules, nucleic acid sequences or sequence segments that have at least 60% homology with the nucleic acid molecules indicated by the described SEQ ID NOs (for example, at least 60% homology includes At least 70% homology, preferably at least 80% homology, more preferably at least 90% homology, even more preferably at least 95% homology, eg at least 98% homology, most preferably at least 99% Homology).
Such mutant molecules may be splice forms from other species or homologous molecules. It will be understood that these variant nucleic acid molecules must nevertheless encode an amino acid sequence having the indicated function. That is, the sequence encoded by the variant of SEQ ID NO: 1 must be an ER signal sequence; the sequence encoded by the variant of SEQ ID NO: 3 must be a linker; and the variant of SEQ ID NO: 5 The sequence encoded by the body must encode a glycoprotein having the function of VSV-G described herein above.

In the present invention, the term “at least% homologous” with respect to a nucleic acid molecule refers to 2 as compared to the number of nucleic acid residues constituting the full length of the amino acid sequence (or all of the parts thereof to be compared). Represents the number of identical nucleic acid matches (“hits”) in one or more aligned nucleic acid sequences. Measured using sequence comparison algorithms known in the art, or manually aligned and visually examined in other conditions using a single row alignment for two or more sequences or subsequences The percentage of nucleic acid residues that are identical (eg, at least 60% homology) when (sub) sequences are compared and aligned for maximum correspondence on the window to be compared or on a specified region ) May be determined. Preferred nucleic acids in the present invention are those in which the described homology exists over a region that is at least 100-150 nucleotides in length, more preferably over a region that is at least 200-400 nucleotides in length. is there. More preferred nucleic acids in the present invention are those having sequence homology over the full length of the nucleic acid molecules described in (a) and (b) above.
In one embodiment, when two sequences are aligned in a row, at least 60, without any sequence extending over the other sequence at the same length, ie, 3 ′ or 5 ′ end. % Homology. Alternatively, if such an extension occurs, it is preferred that no more than 30 nucleotides extend on either end of the other sequence, more preferably no more than 15 nucleotides.

Methods for determining percent sequence homology between sequences, such as the CLUSTALW computer program (Thompson, Nucl. Acids Res. 2 (1994), 4673-4680) or FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci., 1988) , 85; 2444) and other methods using algorithms are well known in the art. The FASTA algorithm typically does not consider internal mismatch deletions or additions in the sequence, in other words, deletions, in its calculation, but this is done manually to avoid overestimating% sequence homology. Can be corrected. However, CLUSTALW takes into account missing sequences in its homology calculations. BLAST and BLAST 2.0 algorithms are also available to those skilled in the art (Altschul, Nucl. Acids Res., 1977, 25: 3389). The BLASTN program for nucleic acid sequences uses word length (W) 11, expectation (E) 10, M = 5, N = 4, and a comparison of both strands by default. For amino acid sequences, the BLASTP program uses word length (W) 3 and expected value (E) 10 as defaults. BLOSUM62 scoring matrix (Henikoff, Proc. Natl. Acad. Sci., 1989, 89: 10915) is an alignment (B) 50, expectation (E) 10, M = 5, N = 4, and for both chains. Use comparison. All these programs may be used for the purposes of the present invention. However, preferably a BLAST program is used.
Thus, all nucleic acid molecules having a predetermined function and having at least 60% sequence homology as determined using any of the above programs or programs available to those skilled in the art, preferably using the BLAST program, It is included in the scope of the present invention.

In a more preferred embodiment of the nucleic acid molecule of the invention, (a) the first sequence segment encoding the ER signal sequence comprises or consists of the nucleic acid sequence shown in SEQ ID NO: 1; (b) encodes a linker The third sequence segment comprises or consists of the nucleic acid sequence shown in SEQ ID NO: 3; and / or (c) the fourth sequence segment encoding said VSV-G is the nucleic acid shown in SEQ ID NO: 5 Contains or consists of a sequence.
In an even more preferred embodiment of the nucleic acid molecule of the invention, the nucleic acid molecule has the full length nucleic acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9.

The invention further relates to a vector comprising the nucleic acid molecule of the invention.
Preferably, the vector is a plasmid, cosmid, virus, bacteriophage, or other vector conventionally used in genetic engineering, for example. The nucleic acid molecules of the invention may be inserted into several commercially available vectors suitable for expression of eukaryotic proteins. Non-limiting examples include prokaryotic plasmid vectors, specifically pUC series, pBluescript (Stratagene), pET series expression vectors (Novagen) or pCRTOPO (Invitrogen), and pREP (Invitrogen). ), PcDNA3 (Invitrogen), pCEP4 (Invitrogen), pMCIneo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2neo, pBPV-1, pdBPVMMTneo, pRSVgpt, pRSVdfp , PlZD35, pLXIN, pSIR (Clontech), pIRES-EGFP (Clontech), pEAK-10 (Edge Biosystems), pTriEx-Hygro (Novagen) and pCINEo (Promega) It is included if the vector.

The nucleic acid molecules of the present invention described above may also be inserted into a vector so that translational fusions with other polynucleotides are generated. Other polynucleotides may, for example, encode proteins that can increase solubility and / or facilitate purification of the fusion protein. Non-limiting examples include pET32, pET41, and pET43.
For vector modification techniques, see Sambrook and Russell "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, NY (2001). In general, a vector comprises one or more origins of replication (ori) and a genetic system for cloning or expression, markers for selection in one or more hosts, such as antibiotic resistance, and one or Further expression cassettes can be included. Suitable origins of replication (ori) include, for example, ColE1, SV40 virus and M13 origins of replication.

  Coding sequences inserted into the vector can be synthesized, for example, by standard methods, or isolated from natural products. Ligation of coding sequences to transcriptional regulatory elements and / or other amino acid coding sequences can be performed using established methods. Transcriptional regulatory elements (part of the expression cassette) that ensure the expression of the coding sequence are well known to those skilled in the art. These elements include regulatory sequences that ensure transcription initiation (eg, translation initiation codon, promoter, enhancer, and / or insulator), internal ribosome entry site (IRES) (Owens, Proc. Natl. Acad. Sci. USA). 98 (2001), 1471-1476), and, if necessary, a poly A signal that ensures termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional and translational enhancers and / or naturally associated promoter regions or heterologous promoter regions. Preferably, the nucleic acid molecules of the invention are operably linked to expression control sequences that allow their expression. The vector may further comprise a nucleotide sequence encoding a secretion signal as an additional regulatory element. Such sequences are well known to those skilled in the art. Furthermore, depending on the expression system used, a leader sequence capable of directing the polypeptide expressed in the intracellular compartment may be added to the coding sequence of the polynucleotide of the present invention. Such leader sequences are well known in the art.

  Possible examples of regulatory elements that ensure transcription initiation include cytomegalovirus (CMV) promoter, SV40 promoter, RSV (rous sarcoma virus) promoter, lacZ promoter, gai10 promoter, human elongation factor 1α-promoter, CMV enhancer CaM kinase promoter, Autographa california multiple nuclear polyhedrosis virus (AcMNPV) polyhedrin promoter or SV40 enhancer. A number of promoters have been described for expression in prokaryotes, including for example the tac-lac promoter, lacUV5 or trp promoter. Examples of additional regulatory elements in prokaryotic and eukaryotic cells include SV40-polyA sites or tk-polyA sites, or transcription termination signals downstream of polynucleotides such as SV40, lacZ and AcMNPV polyhedrin polyadenylation signals. Is included.

Furthermore, the vector of the present invention preferably contains a selection marker. Examples of selectable markers include neomycin resistance, ampicillin resistance and hygromycin resistance. Specially designed vectors allow DNA to reciprocate between different hosts such as bacteria-fungal cells or bacteria-animal cells.
The expression vectors of the present invention can direct the replication and expression of the nucleic acid molecules of the present invention and the encoded fusion proteins. Suitable expression vectors containing the described regulatory elements are known in the art, such as pGreenPuro (System Biosciences, Mountain View, Calif., USA), pRc / CMV, pcDNAI, pcDNA3 (Invitrogene, in particular attached) And prokaryotic expression vectors such as pSPORT1 (GIBCO BRL) or pGEMHE (Promega) or lambda gt11, pJOE, pBR1-MCS series.
The nucleic acid molecules of the invention described herein above can be designed to be introduced directly into cells or via liposomes, phage vectors or viral vectors (eg, adenoviruses, retroviruses). Also good. Furthermore, as a eukaryotic expression system for the nucleic acid molecules of the present invention, a baculovirus system or a system based on vaccinia virus or Semliki Forest virus can be used.

The invention further relates to a host cell comprising a nucleic acid molecule or vector of the invention.
Suitable prokaryotic hosts include, for example, E. coli, Streptomyces, Salmonella or Bacillus bacteria. Suitable eukaryotic host cells are, for example, yeasts such as Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, or chicken cells such as DT40 cells. Insect cells suitable for expression are, for example, Drosophila S2 cells, Drosophila Kc cells, or Spodoptera Sf9 cells and Sf21 cells. Suitable zebrafish cell lines include but are not limited to ZFL, SJD or ZF4.
Mammalian host cells that can be used are human Hela, HEK293, HEK293T, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, COS1, COS7 and CV1, quail QC1-3 cells, mouse L cells, mouse sarcoma cells, bovine melanoma cells Human CAP or CAP-T cells and Chinese hamster ovary (CHO) cells. Also within the scope of the invention are primary mammalian cells or cell lines. Primary cells are cells obtained directly from an organism. Suitable primary cells are, for example, mouse embryonic fibroblasts (MEF), mouse primary hepatocytes, cardiomyocytes, nerve cells, and mouse muscle stem cells (satellite cells), human skin fibroblasts and human lung fibroblasts, human Epithelial cells (nose, trachea, kidney, placenta, intestine, bronchial epithelial cells), human secretory cells (derived from salivary glands, sebaceous glands, sweat glands), human endocrine cells (thyroid cells), human adipocytes, human smooth muscle cells, human skeleton Muscle cells and stable immortal cell lines derived from them (eg, hTERT or oncogene immortalized cells).

Appropriate culture media and conditions for such host cells are known in the art.
The host cell in this embodiment can be, for example, a method for amplification of a vector of the invention, a method for production of a fusion protein of the invention, or direct production of lentiviral particles, as detailed later herein. Can be used in a method for

The present invention further relates to polypeptides encoded by the nucleic acid molecules of the present invention.
The polypeptide of the present invention may have the entire amino acid sequence selected from the sequence represented by SEQ ID NO: 8 or SEQ ID NO: 10, for example.

The present invention further relates to a method for producing the VSV-G fusion protein of the present invention, which comprises culturing the host cell of the present invention under suitable conditions and isolating the produced VSV-G fusion protein. To a method comprising:
Suitable conditions for culturing prokaryotic or eukaryotic hosts are well known to those skilled in the art. For example, a suitable condition for culturing bacteria is culturing under aeration in Luria Bertani (LB) medium. In order to increase the yield and solubility of the expression product, the medium can be buffered or supplemented with suitable additives known to improve or promote yield and solubility. E. coli can be cultured at 4 ° C. to about 37 ° C., the exact temperature or continuous temperature of which depends on the overexpressed molecule. In general, those skilled in the art will also recognize that these conditions may have to meet host requirements and expressed protein requirements. If the inducible promoter controls the nucleic acid of the invention in a vector present in the host cell, the expression of the polypeptide can be induced by the addition of a suitable inducing agent. Appropriate expression protocols and strategies are known to those skilled in the art.

Depending on the cell type and its special requirements, mammalian cell culture is, for example, RPMI medium containing 10% (v / v) FCS, 2 mM L-glutamine and 100 U / ml penicillin / streptomycin, Can be performed in Williams'E medium or DMEM medium. Cells can be stored, for example, at 37 ° C. in a 5% CO ₂ , water-saturated atmosphere, or 41 ° C. for DT40 chicken cells. A suitable medium for insect cell culture is, for example, TNM + 10% FCS or SF900 medium. Insect cells are usually cultured at 27 ° C. as adherent culture or suspension culture. Suitable expression protocols for eukaryotic or vertebrate cells are well known to those skilled in the art and can be derived, for example, from Sambrook and Russel, cited above.

The term “isolation” refers to the selective collection of manufactured VSV-G fusion protein by removing the VSV-G fusion protein produced from the host cell or from the medium in which the host cell was cultured. . Preferably, the isolated VSV-G fusion protein is 100% pure, i.e. does not contain any other components that are not the VSV-G fusion protein of the invention. Methods for isolating the produced fusion proteins are well known in the art and include ion exchange chromatography, gel filtration chromatography (size exclusion chromatography), affinity chromatography, high pressure liquid chromatography (HPLC), reverse Method steps such as phase HPLC, disc gel electrophoresis or immunoprecipitation are included, for example, but not limited to, Sambrook and Russel cited above.
It will be appreciated by those skilled in the art that the term “isolation of the produced fusion protein” refers to the isolation of the protein encoded by the nucleic acid molecule of the present invention.

  The invention further comprises (a) a VSV-G fusion protein domain encoded by a nucleic acid molecule of the invention; and (b) a VSV-G not linked to a (poly) peptide comprising and / or consisting of a cell membrane binding domain. To lentiviral vector particles pseudotyped with

  A “lentiviral vector particle”, also referred to herein as a “lentiviral vector”, is a lentiviral particle, a vector based on a subclass of retrovirus that can be integrated into the genome of a non-dividing target cell. A unique feature of lentiviruses is that they have a self-inactivating (SIN) region of replication in contrast to other retroviral vectors. Lentiviruses are well known in the art, eg, Retroviruses, Coffin JM, Hughes SH, Varmus HE, Cold Spring Harbor (NY): Cold Sprinh Harbor Laboratory Press; 1997; ISBN-10: 0-87969- 571-4; O'Connell RM, Balazs AB, Rao DS, Kivork C, Yang L, Baltimore D. Lentiviral vector delivery of human interleukin-7 (hIL-7) to human immune system (HIS) mice expands T lymphocyte populations. PLoS One. 2010 Aug 6; 5 (8): e12009; Matrai J, Chuah MK, VandenDriessche T. Recent advances in lentiviral vector development and applications.Mol Ther. 2010 Mar; 18 (3): 477-90 Has been.

  Lentiviral vector particles are based on, for example, lentiviruses of the group of cattle, horses, cats, sheep / goats, or primate lentiviruses. Preferably, the lentiviral vector is based on a primate lentivirus such as HIV1, HIV2 or SIV virus. Most preferably, the lentiviral vector is based on the HIV1 lentivirus. As one skilled in the art knows, most (commercially) lentiviral vectors represent a mixture of viral components derived from different viruses and are therefore to some extent “hybrid” vectors. For example, the lentiviral vector may contain components derived from HIV1, VSVg, CMV, WPRE virus. Such hybrid vectors are clearly recognized by the present invention.

  The term “pseudotype” as used herein in the context of viral vectors refers to the regulation of the cell type specificity of viral vectors by the incorporation of foreign viral envelope proteins. This approach is well known in the art and is described, for example, in Bischf et al. (Flexibility in cell targeting by pseudotyping lentiviral vectors. Methods Mol Biol. 2010; 614: 53-68). This approach can be used to alter the tropism of the host and / or reduce or increase the stability of the virus. For example, the use of VSV-G for lentiviral pseudotyping is described, for example, by Burns et al. (Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad. Sci USA. 1993; 90 (17): 8033-8037).

  The term “VSV-G not linked to a (poly) peptide comprising or consisting of a cell membrane binding domain” is also referred to herein as wild-type VSV-G. In this context, the term “wild type” refers only to the fact that the cell membrane binding domain is not fused to the VSV-G used. However, the use of non-natural VSV-G molecules is not excluded unless the non-natural (ie, modified) VSV-G molecule is linked to a (poly) peptide that comprises or consists of a cell membrane binding domain.

  As used herein, the terms “VSV-G fusion protein” and “VSV-G not linked to...” Are not limited to “one” of the described molecules. Instead, these terms define the type of molecule present without any particular limitation on the number. In other words, the term “VSV-G fusion protein” explicitly encompasses one or more VSV-G fusion proteins, and does the term “VSV-G not linked to ...” contain a cell membrane binding domain? Or explicitly includes one or more VSV-Gs not linked to a (poly) peptide consisting of.

  In accordance with the present invention, lentiviral vector particles pseudotyped with two different types of VSV-G protein, ie, lentiviral vector particles pseudotyped with the fusion protein of the invention described hereinabove (optional (a )) And lentiviral vector particles pseudo-typed with wild type (wt) VSV-G (option (b)) that are not fused to the cell membrane binding domain. In other words, each individual lentiviral vector particle expresses both a modified VSV-G glycoprotein and a wild type VSV-G glycoprotein on its surface.

  In accordance with the present invention, it has surprisingly been found that pseudotyping with both of these VSV-Gs is advantageous over using only one of these proteins. First, the use of 100% of the VSV-G fusion protein of the present invention is unable to infect cells in a cytometric assay, as shown, for example, in Example 4, and thus the intended transduction. Not achieved. Secondly, as shown in the appended examples, the mixture of the molecule of the present invention and the wild type molecule led to an improvement in the infection rate compared to the case of the wild type alone.

In a preferred embodiment of the lentiviral vector particles of the present invention, the ratio of (a) :( b) indicated by said pseudotyped lentiviral vector particles of the viral envelope is 10%: 90% and 50% : Between 50%.
In other words, according to this preferred embodiment, the ratio of the VSV-G fusion protein of the present invention to wt-VSV-G is about 10% of the VSV-G fusion protein of the present invention to about 90% wt-VSV-G. And about 50% of the VSV-G fusion protein of the invention versus about 50% wt-wt-VSV-G. Preferably, the ratio is about 33% VSV-G fusion protein of the present invention and about 67% wt-VSV-G.

  The term “about” as used herein includes explicitly stated values and small errors therefrom. In other words, a percentage of “about 33%” includes 33% of the stated amount, but need not be exactly as it is, and may vary by a few percent, and thus, for example, 31%, 32%, 34 % Or 35%. Those skilled in the art will recognize that such values are relative values that do not require complete accuracy, as long as they roughly correspond to the stated values. Thus, from the stated values, for example, an error of 15%, more preferably an error of 10%, and most preferably an error of 5% is included in the term “about”.

  The present invention further provides a method for producing the pseudotyped lentiviral vector particles of the present invention, comprising the steps of efficiently producing and packaging a nucleic acid comprising (i) a viral particle protein and an LTR in a host cell. One or more packaging plasmids encoding accessory proteins necessary for (i) a vector comprising a nucleic acid molecule of the invention; and (iii) a (poly) peptide comprising or consisting of a cell membrane binding domain. A method comprising transfecting a vector comprising a nucleic acid molecule encoding non-VSV-G.

  Methods for producing such pseudotyped lentiviral vectors are well known in the art and are described, for example, in Naldini, L. (1998) [61]. Host cells, preferably HEK293, HEK293T, CAP or CAP-T cells are used. A number of vectors are transfected into these cells, including, for example, packaging vectors encoding viral proteins such as capsids, reverse transcriptase, and vectors carrying nucleic acid molecules additionally introduced into pseudotyped lentiviral vector particles. Or electroporated. Alternatively, nucleofection is used for introduction of the vector. In addition, additional vectors containing genetic material delivered by pseudotyped lentiviral vector particles may be transfected.

These vectors can be obtained by direct introduction or by electroporation (eg, using Multiporator (Eppendorf), Genepulser (BioRad), MaxCyte transfection system (Maxcyte)), PEI (Polysciences, Warrington, Eppelheim), Introduction into host cells via Ca ²⁺ mediated transfection, liposomes (eg “Lipofectamine” (Invitrogen)), or non-liposomal compounds (eg “Fugene” (Roche) or nucleofection (Lonza)) Can be introduced.

  All of the definitions and preferred embodiments described herein above for the pseudotyped lentiviral vector particles of the invention apply mutatis mutandis to the method of producing the pseudotyped lentiviral vector particles of the invention. For example, the preferred ratio between the VSV-G fusion protein of the present invention and VSV-G not fused to the cell membrane binding domain is equally applicable to the ratio used in the method for producing pseudotyped lentiviral vector particles of the present invention. Thus, for example, to obtain a preferred ratio of VSV-G that is not linked to a (poly) peptide comprising or consisting of about 67% cell membrane binding domain and about 33% VSV-G fusion protein of the invention, Adding twice the option (iii) vector to the host cell compared to the option (ii) vector results in a ratio of about 2: 1, ie 67% to 33%.

  The present invention further provides a method for transducing a cell, the method comprising contacting a cell to be transduced with the pseudotyped lentiviral vector particle of the present invention under conditions suitable for transduction, to the cell. It relates to a method comprising a transducing step. Accordingly, the present invention also relates to the use of the lentiviral vector particles of the present invention for transduction of target cells.

  The term “transducing” as used herein is well known in the art, and is through the process of introducing genetic material into a cell, and optionally via viral vector particles. Refers to the process of integrating into the cell's genome. Does the genetic material comprise viral RNA in combination with one or more target RNA sequences (hereinafter referred to as target sequences) contained in the viral vector particle intended for integration into the genome of the target cell? Or consist of.

  The term “contact” as used herein in the context of this method of the invention refers to a transduced cell (hereinafter also referred to as “target cell”) as a retroviral vector so that a transduction event can occur. Refers to contact. The contact conditions that cause a transduction event are well known in the art and may depend to some extent on the cells to be transduced. For example, some target cells are more difficult to transfect than others and may need to be transferred into a specific medium before transduction with a viral vector is achieved. Corresponding methods and conditions are described in, for example, Jacome et al. (Lentiviral-mediated Genetic Correction of Hematopoietic and Mesenchymal Progenitor Cells From Fanconi Anemia Patients. Mol Ther. 2009 June; 17 (6): 1083-1092), Chu et al. (Efficient and Stable Gene Expression into Human Osteoclasts Using an HIV-1-Based Lentiviral Vector. DNA Cell Biol. 2008 June; 27 (6): 315-320) or Poczobutt et al. (Benign mammary epithelial cells enhance the transformed phenotype of human breast cancer cells. BMC Cancer. 2010; 10: 373). Typical conditions are described in the examples.

  In accordance with the present invention, the cell to be transduced can be any cell of interest targeted for transduction with a viral vector. The term “cells / cells” as used in the present invention refers to single cells and / or isolated cells, or cells that are part of a multicellular entity such as a tissue, organism or cell culture. In other words, the method can be performed in vivo, ex vivo or in vitro. Preferably, the transduced cell is a eukaryotic cell, including any cell of a multicellular eukaryote, preferably a cell derived from an animal such as a vertebrate. More preferably, the transduced cell is a mammalian cell. According to the method of the present invention, cells of different mammalian subclasses such as protozoa or veterinary subclasses may be used depending on the specific purpose to be achieved through modification by transducing the genome of the mammalian cell. . For example, within subclasses of the subclass of animals, preferably cells of the lower class of the subclasses are more preferably cells of animals of other primates, artiodactylae, teratoids, rodents and rabbits. Used in the method of the present invention. Furthermore, within one species, based on the type of tissue and / or ability to differentiate, depending on the purpose achieved by modifying the genome via transduction into the target cell according to the method of the invention, Cells used in the method of the present invention can be similarly selected. Three basic cell categories: germ cells, somatic cells and stem cells can in principle be transduced by the method of the present invention, but these constitute the mammalian body. Germ cells are cells that give birth to gametes and therefore continue through generations. Stem cells not only divide and differentiate into various specialized cell types, but can also regenerate themselves to produce more stem cells. There are two main types of stem cells in mammals: embryonic stem cells and adult stem cells. Somatic cells include all cells that are not gametes, germ cells and undifferentiated stem cells. Mammalian cells can be classified by their ability to differentiate. Totipotent (also known as totipotent) cells are cells that can differentiate into all cell types of adult organisms, including zygotes (fertilized eggs) and subsequent placental tissues such as blastomeres. On the other hand, pluripotent cells such as embryonic stem cells cannot contribute to extraembryonic tissues such as placenta, and have the ability to differentiate into one of three germ layers: endoderm, mesoderm, and ectoderm. Pluripotent progenitor cells have the ability to yield multiple cells with a limited number of cell lines. Furthermore, there are few pluripotent cells that can develop into only a few cell types, and unipotent cells (sometimes called progenitor cells) that can develop into only one cell type. There are four basic types of tissues: muscle tissue, nerve tissue, connective tissue and epithelial tissue, from which cells used in the method of the invention such as lymphoid cells or neural stem cells can be derived. To the extent that human cells are envisaged for use in the methods of the present invention, it is preferred that human cells have not been obtained from human embryos, particularly by methods that involve the destruction of human embryos. On the other hand, human embryonic stem cells are commercially available or can be freely disposed of by those skilled in the art to be collected from existing embryonic stem cell lines obtained by methods that do not require destruction of human embryos. it can. Alternatively, or in place of human embryonic stem cells, pluripotent cells similar to embryonic stem cells, such as induced pluripotent stem (iPS) cells, can be used and their production is at the level of the art. (Hargus G et al., 2010, Proc Natl Acad Sci USA, 107: 15921-15926; Jaenisch R. and Young R., 2008, Cell 132: 567-582; Saha K, and Jaenisch R., 2009, Cell Stem Cell 5: 584-595).

As described hereinabove and as shown in the appended examples, even cells that are difficult to transduce, such as lymphoma cell lines or tumor cell lines, are transduced by the method using lentiviral vector particles of the present invention. It has been found according to the present invention to be able to.
In a preferred embodiment of the method of transducing cells of the invention, the method further comprises contacting the cell with an adjuvant, preferably a poloxamer having a molecular weight of 12.8 kDa to about 15 kDa.
The term “adjuvant” as used herein relates to a compound that increases the efficiency of lentiviral transduction. Non-limiting examples of adjuvants include, for example, poloxamers having a molecular weight of 12.8 kDa to about 15 kDa, and polybrene.
Preferably, the method of transducing cells of the invention further comprises contacting said cells with a poloxamer having a molecular weight of 12.8 kDa to about 15 kDa.

The term “poloxamer” is well known in the art and is a nonionic triblock copolymer composed of a central polyoxypropylene hydrophobic chain flanked by two polyoxyethylene hydrophilic chains. Refers to a polymer. The block copolymer can be represented by the following formula:
_{HO (C 2 H 4 O)} x (C 3 H 6 O) z (C 2 H 4 O) yH
(Wherein z is an integer such that the hydrophobic group represented by (C ₃ H ₆ O) has a molecular weight of at least 2250 Da. X or y is an integer of about 8 to 180 or more. .)
Poloxamers are also known under the trade names “Pluronic®” or “Synperonic®” (BASF). The length of the polymer block can be customized, so that there are many different poloxamers. The poloxamer used according to the method of the present invention is a poloxamer having a molecular weight of at least 12.8 kDa to about 15 kDa. As is apparent from the above general formula, the poloxamer having the corresponding molecular weight can be constituted by changing the length of the polymer block constituting the poloxamer. For example, two poloxamers can have approximately the same molecular weight but structurally different. Because one poloxamer has more hydrophobic block polymer repeats and fewer hydrophilic block polymer repeats, the other poloxamer has more hydrophilic block polymer repeats and less hydrophobic This is because the block polymer may have repetition. For example, z can range from 42 to 52, such as at least (as each value) 43, 44, 45, 46, 47, 48, 49, 50 or at least 52, and x + y is at least ( Each value can be in the range of 220-360, such as 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340 or at least 350. Preferably, z is in the range of 44-50 and x + y is in the range of 235-266. Since the synthesis of block copolymers is not precise, the above values may not be accurately achieved by synthesis, and their average values will vary somewhat (as described herein). Preferably, the poloxamer has a molecular weight of 12.8 to about 14.9 kDa, a molecular weight of about 13.2 to about 14.9 kDa, a molecular weight of about 13.4 to about 14.9 kDa, more preferably about 14.0 to about It has a molecular weight of 14.9 kDa, a molecular weight of about 14.3 to about 14.8 kDa, a molecular weight of about 14.5 to about 14.7 kDa, and most preferably a molecular weight of about 14.6 kDa. As will be appreciated by those skilled in the art, the method can be performed using a number of poloxamers. Thus, unless otherwise specified, the term “poloxamer” as used herein may be used interchangeably with the term “poloxamers” (representing a collection of poloxamers, also referred to as a poloxamer mixture). As outlined herein, the synthesis of poloxamers is not precise and results in poloxamer mixtures of various molecular weights. Thus, as used herein, the term “average” related to the molecular weight of poloxamer (s) is a result of the lack of technical ability to produce poloxamers that all have the same composition, and thus the same molecular weight. Used. Accordingly, poloxamers produced according to methods in the art exist as a mixture of poloxamers, each having diversity with respect to its molecular weight, but the mixture as a whole has the average molecular weight specified herein. Those skilled in the art are in a position to obtain poloxamers that can be used in the methods of the invention. For example, BASF and Sigma-Aldrich provide poloxamers as described herein. Methods for measuring molecular weight are well known in the art and are described in standard textbooks on chemistry. Experimentally high pressure liquid chromatography (HPLC) can be used to determine the molecular weight of the poloxamer.

  Lentiviral vector particles and an adjuvant, preferably poloxamer, can be added to the target cells at the same time, eg, as a mixture or sequentially, so long as both compounds are in contact with the target cells at the same time to allow transduction. . Preferably, the target cells, lentiviral vector particles and adjuvant (poloxamer) are at least 5 hours, such as at least 6 hours, at least 7 hours, at least 8 hours, more preferably 9 hours, at least 10 hours, at least 11 hours, most preferably Is in contact for at least 12 hours. Also envisioned are longer times, eg, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, or at least 24 hours. Preference is given to the simultaneous addition of lentiviral vector particles and an adjuvant, preferably poloxamer.

The use of the poloxamer described herein above has significantly increased transduction rates of retroviral vectors in attached suspended target cells, essentially without affecting the viability of the target cells. (WO2013 / 127964), found. Briefly, when a lentiviral vector is used, “Synperonic® F108” (HO— [CH ₂ CH ₂ O] x— [CH ₂ C ₂ H ₄ O] z— [CH ₂ CH ₂ O ] Y (x + y = 265.45 and z = 50.34 (Kabanov, A., Zhu, J. & Alakhov, V. Adv. Genet. 53, 231-261 (2005)); average molecular weight: 14.6 kDa) Poloxamer, referred to as cells that are lower than polybrene, even at a concentration 100 times higher than the state-of-the-art transduction enhancer polybrene (1,5-dimethyl-1,5-diazaundecamethyl polymethobromide) Showed toxicity and high transformation efficiency (HEK293T cells) Most surprisingly, the transfection effect of the poloxamer used was not limited to a particular cell type Many established tumor cells Stock feel Synperonic® F108 (an average molecular weight of 14.6 kDa consisting of 265 hydrophilic ethylene oxide (EO) units and 50 hydrophobic propylene oxide (PO) units), which was difficult to dye; It has been shown in WO 2013/127964 that the use of (defined) significantly increased the infection rate of lymphoma cell lines that were difficult to transfect.

In a preferred embodiment of the method of the invention, the poloxamer has the formula:
_{HO- [CH 2 CH 2 O]} x- [CH 2 C 2 H 4 O] z- [CH 2 CH 2 O] y
(Where x + y = 265.45, and z = 50.34 (mean value (Synperonic® F108)))
Or the poloxamer has the formula:
_{HO- [CH 2 CH 2 O]} x- [CH 2 C 2 H 4 O] z- [CH 2 CH 2 O] y
(Wherein, x + y = 236.36 and z = 44.83 (average value (F98)))
Have

  The first formula, poloxamer, is known in the art as Synperonic® F108 and consists of about 265 hydrophilic ethylene oxide (EO) units and about 50 hydrophobic propylene oxide (PO) units. Synthesized as white granules having an average molecular weight of .6 kDa. The block copolymer is synthesized by continuously adding PO monomer and EO monomer in the presence of an alkaline catalyst. This synthesis begins with polymerization of the PO block, followed by extension of the EO chain at both ends of the PO block. Since the synthesis of the block copolymer is not precise, the repetition of x + y and z is shown as an average value. Thus, and with respect to the term “mean value”, the above definition of Synperonic® F108 also includes poloxamers that deviate from the median, ie, poloxamers that fall within the standard deviation from the mean (mean value). This ratio gives a particularly satisfactory solubility in water or phosphate buffer (Kabanov, A., Zhu, J. & Alakhov, V. Adv. Genet. 53, 231-261 (2005)). In aqueous solution, it is described that a single poloxamer molecule called a unimer self-assembles as micelles with a PO core and an EO shell. Synperonic® F108 as an exemplary poloxamer having a molecular weight within the range defined herein is a trait in target cells, particularly in target cells that are known to be difficult to transfect. It is shown in WO2013 / 127964 that it acts as a powerful increaser of the introduction effect.

A poloxamer of the second formula is known in the art as F98 (Kabanov, A et al. Advances in Genetics. 2005; 53: 231-261) and as Synperonic® F108 according to the present invention. Preferably used. The definition for the term “mean value” given to Synperonic® F108 also applies to F98.
In a preferred embodiment of the method of the present invention, the poloxamer is provided at a concentration of about 50-5000 μg / ml.
The term “about” as defined herein applies to the embodiments mutatis mutandis.

Particularly preferred is a concentration of about 100-4000 μg / ml, more preferably about 200-3000 μg / ml, even more preferably about 300-2000 μg / ml, even more preferably about 400-1500 μg / ml, and most The concentration is preferably about 450 to 1250 μg / ml. Also preferred are concentrations of about 600-1000 μg / ml, 700-1000 μg / ml, 800-1000 μg / ml, 900-1000 μg / ml. At the latter concentration, the poloxamer as defined herein is in the liquid state when diluted in water or phosphate buffer. As will be appreciated by those skilled in the art, transducing cells with liquid poloxamers can be more practical and more convenient because, for example, convenient handling such as simpler pipetting is possible.
According to this method of the invention, the poloxamer is preferably dissolved in water, phosphate buffer, or directly in cell culture medium. Poloxamers can be dissolved, for example, in water or phosphate buffer to obtain a 100 mg / ml stock solution that can be diluted to a predetermined working concentration. A poloxamer solution having a concentration of 200 mg / ml or more is in a gel form. At a concentration of 200 mg / ml or less, the poloxamer solution is in a liquid state. Preferably, the concentration is such that the poloxamer is provided in a liquid state.

In a further preferred embodiment of the method of the invention, the target cell is further contacted with one or more polycationic substances selected from the group of polycationic polymers or polycationic peptides.
In the present invention, the “polycationic polymer” refers to a charged polymer in which the repeating unit has a positive charge, and the positive charge of the repeating unit originates from a protonated nitrogen moiety. For example, a group that is positively charged with polyethyleneimine (PEI) is an imine group. Other non-limiting examples of polycationic polymers are polybrene materials (1,5-dimethyl-1,5-diazaundecamethylene polymethobromide, hexadimethrine bromide).
The term “polycationic peptide” refers to a positively charged peptide. For example, poly -L- _{lysine, (C 6 H 12 N 2} O) n ( where, according to the present invention, n at least 2, such as at least 20, preferably 200 and 500, more preferably between 500 and 2500 A homopolymer polycationic peptide having a molecular formula of (but not limited to).

In a further preferred embodiment of the method of transducing cells of the invention, said method comprises introducing pseudotyped lentiviral vector particles into said cells before, simultaneously with or after contacting the target cells with said adjuvant. And a further step of spinoculating.
In an even more preferred embodiment of the method of transducing cells of the present invention, said method comprises the step of: It includes the additional step of spinoculating with the cell.
The term “spinoculation” is well known in the art, and centrifugation of target cells with lentiviral vector particles to ensure close contact for viral particle uptake into cells. About force use inoculation. Spinoculation protocols are well known in the art and are described, for example, for lentiviral vectors in Millington et al., 2009 [44]. The spinoculation step is performed before, simultaneously with, or after contacting the target cells with the adjuvant, preferably the poloxamer. Preferably, the spinoculation step is performed after contacting the target cells with an adjuvant, preferably a poloxamer.
The spinoculation step further increases the transduction rate achieved with the methods of the invention, as shown in Example 5, especially in cells that are difficult to transfect.

In a further preferred embodiment of the method for transducing cells of the invention, the cells to be transduced are selected from the group consisting of tumor cells, lymphoid cells, epithelial cells, neural cells and stem cells, and / or Preferably, the transduced cells are part of a heterogeneous cell population.
The term “tumor cell” in the present invention is well known in the art and refers to a tumor cell involved in the formation of benign, pre-malignant or malignant tumors. Tumor cells that are malignant are commonly referred to as cancer cells and may have the ability to metastasize or spread to adjacent tissues. Preferred tumor cells include, for example, pancreatic tumor cells (eg, AsPC-1 and PANC-1 cells), lymphoma cell lines (eg, KARPAS-299, SUDHL-1, SUP-M2, and SR-786 cells), and Breast cancer cells (for example, MCF7, MDA-MB-361, T47d cells, etc.).
The term “lymphoid cell” refers to cells involved in the production of lymphocytes and the lymphocytes themselves. The term “lymphocyte” refers to small lymphocytes (B lymphocytes, T lymphocytes, plasma cells) and natural killer cells, as is well known in the art. Lymphocyte cells further include, for example, lymphocyte dendritic cells and lymphocyte progenitor cells such as prolymphocytes, lymphoblasts, and lymphocyte common progenitor cells.

The term “epithelial cell” is well known in the art. Epithelial cells cover the entire surface of cavities and structures throughout the body and form many glands. Epithelial tissues can be classified into simple epithelium (1 cell thickness) and stratified epithelium (several cell layers). Epithelial cells are further classified into squamous, cubic, columnar and multi-row epithelial cells according to their morphology. For example, the human stomach and intestines are covered with epithelial cells. In addition, epithelial cell lines also include breast cancer cells (eg, MCF7, MDA-MB-361 and T47D cells) or cells of the cell line HEK293T.
A “neural cell” is well known in the art and is a cell that is electrically excitable and transmits information by electrical and chemical signaling. For example, there are various specialized nerve cells such as sensory neurons and motor neurons. For example, basket cells, Betz cells, medium spiny neurons, Purkinje cells, cone cells, Renshaw cells, granule cells, or anterior horn cells can be used as target cells according to the present invention. A “neural tumor cell” is a tumor cell of neural origin, such as glioma, medulloblastoma, astrocytoma, and other cancers from the nervous system. Glioma cell lines (eg, U87 and LN18) can be used in the methods of the present invention.

The term “stem cell” is well known in the art and is described in detail herein. Preferred stem cells used according to the method of the present invention are, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, cancer stem cells.
All of these cell types are typically considered difficult to transfect or transduce, but can now be transduced more efficiently based on the methods of the present invention.
In additional or preferred embodiments, the transduced cells are part of a heterogeneous cell population, ie, a cell population comprising different types of cells (also referred to herein as a “mixed population”). As shown in the appended examples, in particular in Examples 4 and 6, transduction is clearly enhanced in cells with target molecules to which the cell membrane binding domain can bind. In mixed cell populations containing cells with and without such target molecules, the transduction equilibrium is shifted towards the cells with the target molecule. Thus, it has been shown herein that even in the presence of competing non-target cells, the relative specificity of the retargeted lentiviral particles for those target cells can be obtained.

Furthermore, the present invention is not linked to a (poly) peptide further comprising (a) (i) a nucleic acid molecule of the present invention; or (a) (ii) a nucleic acid molecule of the present invention and a cell membrane binding domain. A nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding VSV-G;
And / or (b) (i) a vector of the invention; or (b) (ii) a vector of the invention and VSV-G not linked to a (poly) peptide comprising or consisting of a cell membrane binding domain A vector comprising a nucleic acid molecule comprising or consisting of a nucleic acid sequence;
And / or (c) (i) a host cell of the invention; or (c) (ii) a host cell of the invention and a VSV-G not linked to a (poly) peptide comprising or consisting of a cell membrane binding domain. A host cell comprising a vector comprising a nucleic acid molecule comprising or consisting of the encoding nucleic acid sequence;
And / or (d) (i) a polypeptide of the present invention; or (d) (ii) a polypeptide of the present invention and a VSV-G not linked to a (poly) peptide comprising or consisting of a cell membrane binding domain;
And / or (e) pseudotyped lentiviral vector particles of the present invention;
In addition, the present invention relates to a kit containing instructions for use as necessary.

  The various components of the kit may be packaged in one or more containers, such as one or more vials. Vials contain, in addition to the above components, preservatives or buffers for storage, media for maintenance and storage, such as ES cell medium, DMEM, MEM, HBSS, PBS, HEPES, hygromycin, puromycin, among others. , Penicillin-streptomycin solution, gentamicin. Advantageously, the kit includes instructions for the use of said components that allow one of ordinary skill in the art, for example, to conveniently carry out the various embodiments of the invention. Any of the above components may be used in an experimental setting.

  The definitions and preferred embodiments described herein relating to a nucleic acid molecule of the invention, a vector of the invention, a host cell of the invention, a polypeptide of the invention, or a pseudotyped lentiviral vector particle of the invention It is applied mutatis mutandis to the components of the kit. For example, pseudotyped lentiviral vector particles of the present invention can be combined with poloxamers as described herein. As another example, the preferred ratio of the fusion protein of the VSV-G of the present invention to the VSV-G not linked to the cell membrane binding domain is the ratio of the components of the kit according to options (a) (ii). Applies equally. Thus, particularly preferably, the kit comprises or consists of a nucleic acid substance according to the invention in an amount of 33% of the total amount of VSV-G and an amount of cell membrane binding domain of 67% of the total amount of VSV-G ( Nucleic acid sequences encoding VSV-G not linked to a poly) peptide are included.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the patent specification, including definitions, will control.
In this specification, and in particular with regard to the embodiments characterized in the claims, each embodiment described in a dependent claim shall have each dependent claim on which that dependent claim is dependent (independent or dependent). It is intended to be combined with each embodiment of the claims. For example, independent claim 1 lists three options A, B and C, dependent claim 2 lists options D, E and F, and claim 3 which is dependent on claims 1 and 2 has option G. , H and I, unless specifically stated otherwise, this specification includes A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I, C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; Should be understood.

Similarly, any combination of subject matter protected by an independent claim and / or dependent claim, if the dependent claim is dependent on preceding claims, does not enumerate options. It is understood that it is considered explicitly disclosed. For example, if there is independent claim 1, dependent claim 2 is dependent on claim 1 and dependent claim 3 is dependent on both claims 1 and 2, the subject matter of claims 3 and 1 Combinations will be disclosed clearly and clearly, as well as combinations of subject matter of claims 3, 2 and 1. Further, if the dependent claim 4 is subordinate to any of claims 1 to 3, the subject matter of claim 4 and claim 1, the subject matter of claim 4, claim 2 and claim 1 The combination of the subject matter of claim 4, claim 3 and claim 1 and the combination of claim 4, claim 3, claim 2 and claim 1 are clearly and clearly disclosed. Become.
The above considerations apply mutatis mutandis to all appended claims.

FIG. 2 shows the design and production of lentiviral particles containing scFv-VSV-G fusion. The VSV-G plasmid was used for the production of lentivirus supplemented with wild type (wt) and scFv. In order to obtain CD30 or EGFR specificity, scFv antibody fragments against either antigen were cloned between the signal sequence (SS) and the protein sequence of VSV-G. For detection purposes, a His tag was fused to the N-terminus. [Abbreviation] VSV-G: Envelope glycoprotein G of vesicular stomatitis virus. His6: His tag consisting of 6 histidine residues. V _H / V _L : variable heavy chain / variable light chain. FIG. 6 shows transduction of EGFR ⁺ HEK293T cells with different ratios of scFv-αEGFR added lentiviral particles. (A) Yields of 6 different copGFP-encoded lentiviral particles produced using packaging plasmids encoding different ratios of wt-VSV-G and scFv-αEGFR-VSV-G. (B) Immunoblot assay of antibody retargeted lentiviral particles (33% scFv-αEGFR-VSV-G, right lane) with His tag (84 kDa). For reference, 100% wt-VSV-G lentiviral particles (left lane) and lentiviral p24 core protein (24 kDa) were provided. (C) FACS analysis of EGFR expression on the surface of HEK293T cells. FIG. 6 shows transduction of EGFR ⁺ HEK293T cells with different ratios of scFv-αEGFR added lentiviral particles. (D) FACS analysis of GFP expression in HEK293T cells transduced with different ratios of scFv-loaded lentiviral particles (as in (a)) at MOI1 (MFI: mean fluorescence intensity). (E) Statistical quantification of performed transduction experiments in HEK293T cells described in (d) (3 different experiments, mean ± SD, p <0.05 with t test). FIG. 5 shows factors affecting transduction of lentivirus retargeted with antibodies. (A) FACS analysis of EGFR expression on the surface of T47D (EGFR ⁺ ) and ZR75 (EGFR ⁻ ) cells. (B + c) Quantification of GFP expression after transduction of T47D (b) and ZR75 (c) cells at MOI1 with different ratio types of copGFP-encoded scFv-added lentiviral particles. (D) Traits in T47D cells cultured with wt and scFv-αEGFR added lentiviral particles (MOI1) carrying a fusion linker between the scFv antibody fragment and VSV-G or not carrying such a fusion linker Quantification of introductory experiments (3 different experiments, mean ± SD, p <0.05 with t test). It is a figure which shows the transduction of CD30 ⁺ lymphoma cell by scFv- (alpha) CD30 addition lentiviral particle. (A) FACS analysis of CD30 expression on the surface of KARPAS-299, SUP-M2, SUDHL1 and HEK293T cells. It is a figure which shows the transduction of CD30 ⁺ lymphoma cell by scFv- (alpha) CD30 addition lentiviral particle. (B + c) Statistical quantification of transduction experiments in the described cells cultured with or without MOI10 or 1 copGFP-encoded lentiviral particles, poloxamer-based adjuvants and scFv-αCD30 loaded VSV-G (3 different experiments, mean ± SD, p <0.05 with t test, p <0.01 with ** t test). It is a figure which shows the transduction of CD30 ⁺ lymphoma cell by scFv- (alpha) CD30 addition lentiviral particle. Statistical quantification of transduction experiments in the described cells cultured with MOI10 or 1 copGFP-encoded lentiviral particles, poloxamer-based adjuvants and scFv-αCD30 loaded VSV-G with or without (d + e) spinoculation (3 different experiments, mean ± SD, p <0.05 with t test, p <0.01 with ** t test). FIG. 6 shows the transduction of ScFv-VSV-G added target cells in a competitive transduction assay in the presence of non-target cells. (A) FACS analysis of CD30 expression on the surface of HL60 leukemia cells. (B) KARPAS-299 and HL60 cells, or (c) ZR75 and T47D cells were mixed in the same number of cells, (left) with wt lentiviral particles, (right) with 33% scFv-VSV-G at MOI1 Transduction was performed with lentiviral particles. Cell fluorescence dot blots (FSC vs. CD30 or EGFR expression) were able to distinguish between two populations: CD30 ^- HL60 cells in (b) and CD30 ⁺ KARPAS-299 cells, or EGFR ^- ZR75 cells in (c). And EGFR ⁺ T47D cells. In the lower dot blot (GFP expression vs. FSC), the gated cell population was analyzed for copGFP expression. The graph represents one experiment for the three replicates performed.

  The examples illustrate the invention.

Example 1: Materials and Methods
Cell line Human fetal kidney HEK293T cells were cultured in DMEM medium supplemented with 10% fetal calf serum (FCS, Pan Biotech, Eidenbach, Germany) and 2 mM glutamine. Undifferentiated large cell lymphoma cell lines KARPAS-299, SUDHL-1 and SUP-M2, and the promyelocytic leukemia cell line HL60 were cultured in RPMI 1640 medium supplemented with 10% FCS and 2 mM glutamine. Epithelial breast tumor cell lines ZR75 and T47D were cultured in RPMI medium containing 10% FCS, 2 mM glutamine and 0.2 U / ml bovine insulin (Cell Application Inc., San Diego, Calif.).

The engineering MfeI restriction site of the scFv-αCD30− / scFv-αEGFR-VSV-G plasmid is the packaging vector pMD2. Added to the SS of VSV-G (Indiana serotype) available in G (eg available from Addgene.org) via error-prone PCR. In order to introduce a His tag at the N-terminus, scFv-αCD30 and scFv-αEGFR cDNAs (these sequences can be ordered from suppliers such as GeneArt®) are used as MfeI-His tag sense primers. (5'-GCGACCAATTGCCATCATCATCATCATCATGCCCAGGTCAAGCTGCAGGAGTGGACTGAACTGGCAAAG; SEQ ID NO: 11), and XhoI site (5'-GTAATCTCGAGCCACCTCCTGAACCGCCTCCCCGTTTGATTTCCAGCTTGGTGCCACACCGAACGTGGCG; GS GS using GG linker, GS linker GS Amplified. When added, the other half of the linker is PCR (using the Agel restriction site V in VSV-G, using the Agel restriction site V in VSV-G, 5'-GTTATCTCGAGCGGAGGCGGTTCAAAGTTCACCATAGTTTTTCCACACAACAAAAGAAACTG) Bound to the N-terminus. Both products were double digested with either MfeI / XhoI or XhoI / Agel to give pMD2. (Linearized with MfeI / Agel digestion). Reinserted into G. FastDigest restriction enzyme was purchased from Fermentas (Vilnius, Lithuania). Oligonucleotides were obtained from Eurofins MWG Operon (Ebelsberg, Germany).

Lentiviral production The lentiviral transduction vector pGreenPuro (System Biosciences, Mountain View, Calif., USA) allows the expression of copGFP controlled by an internal CMV promoter. Replication-deficient lentiviral particles include 8 μg pGreenPuro, 16 μg or 8 μg packaging plasmids pMDLg / pRRE and pRSV. Rev (available from eg Addgene.org) 4 μg of various ratios of pMD2. G, pMD2. Produced by transient cotransfection of HEK293T cells in 10 cm Petri dishes with G scFv-αCD30 or αEGFR. Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) was used for transfection according to the manufacturer's instructions. Viral particles were collected 48 hours after transfection, cell debris was removed by low speed centrifugation, and filtered using a 0.45 μm steric cup filter. The lentiviral supernatant was concentrated by ultrafiltration using an Amicon-20 column (Millipore, Massachusetts, Billerica) as previously described [24]. An aliquot of concentrated lentivirus was stored at -80 ° C. Viral titer (virus particles per ml of concentrated aliquot) was determined according to the company's protocol by QuickTiter ™ lentivirus quantitation p24 ELISA (BioCat, Heidelberg, Germany) using serially diluted aliquots of lentivirus. .

For the preparation of the immunoblot analysis virus protein, 2 μl of concentrated lentiviral solution was added to urea sample buffer (5% sodium dodecyl sulfate (SDS), 8M urea, 200 mM Tris-HCl, 0.1 mM EDTA, 0.03% bromophenol. Blue, 2.5% dithiothreitol, pH 8.0) was denatured by incubation at 95 ° C. for 10 minutes [25]. Samples were fractionated on SDS polyacrylamide gel (14%) and transferred to nitrocellulose membrane (GE Healthcare, Little Chalfont, UK). His-tagged scFv-αCD30-VSV-G or scFv-αEGFR-VSV-G were conjugated with mouse anti-His antibody (clone 13/45/31, Dianova, Hamburg, Germany), followed by anti-mouse antibody. Detection was performed using horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, California, USA). The lentiviral core protein p24 detected with a mouse antibody (BioCat, Heidelberg, Germany) was used as an internal standard for lentiviral proteins. Blotted membranes were developed with the ECL Advanced Western Blot Detection System (GE Healthcare) as recommended by the supplier.

Lentiviral transduction HEK293T, ZR75 and T47D cells (2 × 10 ⁵ cells / well) have 10 μl of poloxamer based chemical adjuvant (LentiBoost ™, Sillion Biotech, Martins, Germany) [23] Or covered with different MOI (lentiviral particles / cell) in 1 ml lentivirus containing medium without. After culturing at 37 ° C. with 5% CO ₂ for 24 hours, the supernatant was replaced with a fresh medium and further cultured for 24 hours.
KARPAS-299, SUDHL-1 or SUP-M2 suspension cells (10 ⁶ cells / well) were resuspended in 1 ml of lentivirus-containing medium. The plate was centrifuged (spinoculation) at 800 g for 90 minutes. After spinoculation, SUDHL-1 cells were washed directly, resuspended in fresh medium and cultured for 48 hours. After centrifugation, KARPAS-299, SUP-M2 and HL60 cells were cultured overnight in 1 ml of lentiviral media, then washed, resuspended in fresh media and cultured for an additional 24 hours. For competition assays, a mixture of 5 × 10 ⁵ KARPAS-299 and 5 × 10 ⁵ HL60 cells or 10 ⁵ T47D and 10 ⁵ ZR75 cells was added to a 1 ml wrench with the poloxamer-based adjuvant LentiBoost ™. Resuspended in virus-containing medium. Suspended cells were centrifuged at 800 g for 90 minutes, cultured overnight, then washed and cultured again for 24 hours.

Cell Fluorescence Analysis After transduction with lentivirus, cells were washed and resuspended in PBS. Using FACSDiva (BD Biosciences, Heidelberg, Germany), 30,000 events were analyzed for forward and side scatter properties and green fluorescence emission at 530 nm. Mean fluorescence intensity (MFI) quantifies the change in fluorescence intensity of transduced cells.
To detect surface expression of CD30 or EGFR, 10 ⁶ cells were washed twice with PBS supplemented with 2% FCS and 100 μl antibody dilution (1:20 in PBS + 2% FCS; FITC-conjugated αCD30 or αEGFR antibody). And isotype control, Dako, Denmark; PE-conjugated αCD30 or αEGFR antibody, BioLegend, San Diego, Calif., USA) for 1 hour on ice. Prior to cytometric detection (FITC: 530 nm; PE: 610 nm), the cells were washed twice and resuspended in PBS + 2% FCS.

Statistical analysis All experiments were performed technically in duplicate and biologically in triplicate. Mean ± standard deviation (SD) values are shown unless otherwise stated. The results obtained were statistically evaluated by t-test using statistical software SigmaPlot (Systat Software, San Jose, Calif.). Statistical significance was considered at the * p <0.05 level.

Example 2: Design and Production of Lentiviral Particles Containing scFv-VSV-G Fusion For the production of VSV-G pseudotyped lentiviral particles, HEK293T cells were transformed into a co-GFP expressing lentiviral expression vector and three packaging plasmids. (One of them encodes VSV-G (pMD2.G)) and was transiently transfected with a mixture of four plasmids. N-terminal His inserted between the signal sequence (SS) and the N-terminus of full-length VSV-G to incorporate a single chain antibody fragment (scFv-αCD30 or scFv-αEGFR) that recognizes CD30 or EGFR, respectively. A novel VSV-G fusion protein was designed that contained a tag followed by a scFv antibody fragment followed by a flexible linker (GGGSGGGSSGGGS) (FIG. 1). In order to determine the optimal lentiviral vector construction, pMD2 encoding either wild type (wt) VSV-G or scFv-αEGFR added VSV-G. Lentiviral particles retargeted with GFP-encoded scFv with different proportions of G plasmid were generated. Particles retargeted with antibodies with 10%, 20%, 33% and 67% scFv-αEGFR-VSV-G yielded comparable yield (quantified by ELISA assay detecting lentiviral core protein p24) (Fig. 2a). The high ratio of scFv-αEGFR-VSV-G encoding plasmid in the production mixture resulted in a low yield compared to the production of wt-VSV-G lentivirus. Incorporation of scFv-αEGFR-VSV-G into lentiviral particles was shown by immunoblotting of scFv-VSV-G pseudotype particles. Although possible accumulation of membrane-bound VSV-G was observed (white clouds), a clear band of His-tagged scFv-αEGFR-VSV-G was detected (FIG. 2b).

Example 3: different ratios of scFv-αEGFR added was retargeted transduced GFP encoding antibodies of EGFR ⁺ HEK293T cells with lentiviral particles particles cytometric analysis in MOI1 (FIG 2C～e) in EGFR ⁺ HEK293T The ability to infect cells was tested. While wt-VSV-G lentiviral particles can infect 24.1% of HEK293T cells, scFv-VSV-G additional lentiviral particles significantly increase the infection rate by a factor of 2.5 (33% scFv-αEGFR-VSV). It was improved to 62.3% with particles having -G. Lentiviral particles retargeted with an antibody carrying 67% scFv-αEGFR-VSV-G had an infection rate of only half (13.4%) of wt-VSV-G lentiviral particles and were homotypic ( 100%) scFv-αEGFR-VSV-G lentiviral particles were not infectious (FIGS. 2d, e).

Example 4: Lentiviral particles displaying the factor scFv-αEGFR-VSV-G affecting the transduction of lentivirus retargeted with antibodies were able to infect EGFR ⁺ HEK293T cells. In order to confirm the specificity of the improved transduction rate, both antigen expressing T47D breast cancer cells and antigen negative ZR75 cells were used as cell models (FIG. 3a). In T47D cells, 33% scFv-αEGFR-VSV-G lentiviral particles have doubled the infection rate (67.5%) compared to wt-VSV-G lentiviral particles (33.7%) It showed the best performance. As described above, production of viruses containing higher plasmid amounts of scFv-added VSV-G (100%) failed to infect EGFR ⁺ cells in the cytometry assay (FIG. 3b). Transduction of EGFR ^- ZR75 cells with 33% scFv-αEGFR-VSV-G lentiviral particles did not increase the transduction rate (FIG. 3c), indicating that the enhancing effect of scFv targeted lentiviral particles Has been demonstrated to correlate with the presence of antigen on the target cell surface.
Both the size of the fusion protein domain and the attachment mechanism are important for the correct folding and function of the recombinant fusion protein. In order to assess the role of the fusion linker (13 amino acids: GGGSGGGSSGGGS), we have scFv-αEGFR-VSV-G without a linker sequence that separates the variable light chain sequence and the VSV-G gene sequence of the scFv-αEGFR antibody fragment. Was designed (see: FIG. 1a). Production of 33% scFv-αEGFR-VSV-G lentiviral particles without a fusion linker resulted in a strong reduction in virus yield compared to production of 33% scFv-αEGFR-VSV-G lentiviral particles (2.5 × 10 ⁸ vs 9.1 × 10 ⁸ virus particles / ml). Particles retargeted with an antibody without a fusion linker failed to increase the transduction rate of T47D cells (FIG. 3d). We have concluded that the linker sequence between the antibody and VSV-G affects the improved function of lentiviral particles retargeted with scFv-VSV-G.

Example 5: Optimized transduction protocol for CD30 ⁺ lymphoma cells A low lentiviral infectious titer (MOI2 or less) is sufficient to transduce the most adherent epithelial cells. However, hematopoietic cells such as suspended lymphoma cells can transduce only a subset of cell populations, even at high lentiviral titers (MOI10). Thus, an optimized infection protocol was developed by combining spinoculation, poloxamer-based chemical adjuvants, and the VSV-G envelope retargeted with scFv. KARPAS-299, SUP-M2 and SUDHL-1 lymphoma cells show high CD30 surface expression levels compared to CD30 ^- HEK293T cells (FIG. 4a). Standard transduction of lymphoma cells with MOI10 resulted in a transduction rate of 20-40% (FIGS. 4b-d). The addition of spinoculation and chemical adjuvants increased lentiviral infection. Furthermore, when 33% of the lentiviral surface was modified with scFv-αCD30-VSV-G, the transduction rate with MOI10 was improved to a level exceeding 90% (in SUDHL-1 cells, compared with wt-VSV-G). 4 times improvement). Even if the titer of lentiviral particles decreased to MOI1, 50% of the lymphoma cells could be transduced using this transduction protocol. Thus, using this optimized system resulted in a 10-fold increase in transduction efficiency compared to previous art protocols. In contrast, CD30 ^- HEK293T cells were not able to benefit from lentiviral surface modifications, clearly indicating the presence of specific antigens. These data correlate with the effect on EGFR ^- ZR75 cells when scFv-αEGFR-VSV-G added lentivirus was used (FIG. 3c). For all tested cell types, the best transduction results were obtained by using lentiviral vectors pseudotyped with 33% scFv-VSV-G.

Example 6: Transduction of target cells by ScFv-VSV-G addition in the presence of non-target cells In a single cell type culture, lentiviral particles retargeted with scFv were treated with wt-VSV-G We were able to transduce lymphoma and epithelial tumor cells more effectively than type vector. In addition, its specificity was tested in two competitive cell transduction experiments. In the first experiment, adherent EGFR ⁺ T47D cells and EGFR ^- ZR75 cells were co-cultured (FIG. 5a). In a second experiment, CD30 ⁺ KARPAS-299 cells were mixed with CD30 ⁻ HL60 promyelocytic lymphoma cells and both cells were cultured in suspension (FIG. 5b, c). Both mixtures were infected with 100% wt-VSV-G lentiviral particles or 33% scFv-αCD30-VSV-G lentiviral particles at MOI1 under optimized conditions (+ spinoculation, + poloxamer-based adjuvant). It was. Each of the cell mixtures using 100% wt-VSV-G pseudotyped lentiviral particles showed similar transduction rates by FACS analysis (in the first experiment, 24.8% transduced ZR75 cells and 27.3% transduced T47D cells; in the second experiment, 37.1% transduced HL-60 cells and 36.3% transduced KARPAS-299 cells). In comparison with the transduction of adherent T47D and ZR75 cells, experiments with lymphoma cell and HL60 cell suspensions generally had lower MFI (average fluorescence intensity) values for GFP expression.
In both cases, when lentiviral particles retargeted with scFv were used, the transduction equilibrium shifted to the antigen-positive cell type in the mixture (61.9% EGFR ⁺ T47D cells, vs. 17 1% transduced EGFR ^- ZR75 cells and 72.2% CD30 ⁺ KARPAS-299 cells vs. 16.5% transduced CD30 ^- HL60 cells). These findings indicate that lentiviral particles retargeted with antibodies have relative specificity for their target cells, even in the presence of competing non-target cells.

Example 7: A new type of lentiviral particle carrying scFv retargeted VSV-G glycoprotein presenting a linker-fused single chain antibody fragment (scFv) against the considered lymphoma tumor antigen CD30 or the broad epithelial tumor antigen EGFR Was developed. In combination with spinoculation and poloxamer-based adjuvants, high transduction rates in antigen positive cells were also achieved with low MOI.
A variety of chemical adjuvants can be used to enhance lentiviral gene transfer, including chemical polymers based on cationic polymers or lipids that neutralize membrane charge [26-28]. For clinical applications, lentiviral transduction protocols are often based on the use of the retroviral transduction enhancer retronectin, a fibronectin derived fragment [29]. Retronectin has been reported to promote the activity of GALV and RD114 pseudotype vectors, but the transduction of VSV-G pseudotype vectors has been reported [22, 30, 31]. Furthermore, the amount of retronectin added to the suspension cells is difficult because of the surface-based activity.

Searching for new adjuvants suitable for clinical protocols for lentiviral vector transduction, the cationic peptide Vectofusin-1® and parents showing improved gene delivery to CD34 ⁺ hematopoietic cells and primary T cells The recently discovered amphiphilic poloxamer Pluronic® F-108 (poly (ethylene glycol) -block-poly (propylene glycol) -block-poly (ethylene glycol) having an average molecular weight of about 14,600) (Sigma Aldrich)) [32, 23]. Poloxamer F108 has a low toxicity level even at high concentrations (> 5 mg / ml) and the FDA drug master file for the same poloxamer 338® as the chemical composition available from BASF (Kolliphor P338®) is there.

  In the past, antibody-retargeting studies have been performed using retroviruses that display antibodies fused to other glycoproteins or by conjugation-based approaches [33-36]. As a powerful glycoprotein from the viewpoint of the stabilization of the particle aggregate and the ability of membrane fusion [37] that can be maintained after the insertion of a new protein domain at the N-terminus of VSV-G by studies based on VSV-G, The advantages of VSV-G have been shown [38, 39]. By adding a collagen-binding domain [11], an antigen-binding ZZ domain [12] or a fibrinogen binding site [13] to the N-terminus of VSV-G, a modified lentiviral particle (some with wt-VSV-G Could be immobilized on a collagen / antibody coated matrix or fibrin hydrogel. Thereafter, the cells cultured on the matrix to which the virus particles were attached were more efficiently transduced up to 5 times. In one study [11], acceptable titers could only be achieved at 30 ° C. by transporting the modified VSV-G through a temperature sensitive membrane during particle manufacture. These methods improve virus particle immobilization, thereby increasing spatially controlled virus uptake when adherent cells are in close contact with immobilized virus particles and polybrene, and It was shown that the specificity of the virus particle itself was not changed.

In order to enhance the selectivity of lentiviral particles, Kaikkonen et al. [40] fused streptavidin to the transmembrane domain of VSV-G on a gp64 pseudotyped envelope to generate αEGFR-avidin-antibody and αCD46-avidin-antibody. Bonding based bonding was induced. This resulted in a transduction rate more than doubled in the lung tumor cell lines, liver tumor cell lines, and ovarian cancer cell lines that had an MOI of 0.2 to 1.2 in the presence of polybrene. However, such non-covalent binding approaches can be limited because biotinylated antibody-adapter tends to dissociate due to biotinidase activity in serum [41].
Dreja and Piechaczyk [14] added an extraneous signal sequence fused to a scFv antibody fragment directed against ubiquitously expressed human MHC-I to the N-terminus (no linker) of full-length VSV-G. . They showed the formation of lentiviral particles retargeted with antibodies and binding to cells, but only achieved low titers and low infectivity to human cells.

They achieved a 5-fold higher selectivity for human cell transduction (MOI not shown) compared to VSV-G lentiviral particles carrying non-binding scFv antibody fragments in the presence of polybrene. did. Our results support these findings because homotypic (100%) scFv-added VSV-G particles did not transduce target cells with MOI1. The antibody fragment may mask the receptor binding site of VSV-G, or spatial interference may inhibit the fusion ability of VSV-G [42, 43].
In this study, we were able to increase the specificity with high transduction rate by producing lentiviral particles presenting a mixture of wt-VSV-G and scFv-added VSV-G. In all experiments, the best results were obtained at a ratio of 33% of scFv-added VSV-G. By combining spinoculation with a poloxamer-based chemical adjuvant, a 4-fold higher transduction of antigen-positive lymphoma cells was obtained even in the presence of non-targeted HL60 cells.

Conclusion The recombinant scFv-VSV-G fusion strategy described herein, and in particular the preferred strategy in the above examples, is easily adapted to different cellular antigens by altering the affinity of the scFv antibody fragment. it can. Significant increases in gene delivery rates were achieved by combining spinoculation and chemical adjuvants in a challenging non-adherent cell model. These are useful for lentivirus applications in industry and medicine.

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Claims (15)

A nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding a vesicular stomatitis virus envelope glycoprotein (VSV-G) linked to a (poly) peptide comprising or consisting of a cell membrane binding domain comprising:
The nucleic acid sequence is in the 5 ′ to 3 ′ direction,
(A) a first sequence segment encoding an endoplasmic reticulum (ER) signal sequence;
(B) a second sequence segment encoding said (poly) peptide comprising or consisting of a cell membrane binding domain;
A nucleic acid molecule comprising (c) a third sequence segment encoding a linker; and (d) a fourth sequence segment encoding said VSV-G. Said (poly) peptide comprising or consisting of a cell membrane binding domain encoded by said second sequence segment is a single chain antibody, a single domain antibody, a V _H H antibody fragment, a VNAR single chain antibody and 2. The nucleic acid molecule of claim 1 selected from the group consisting of protein scaffolds.   The (poly) peptide comprising or consisting of a cell membrane binding domain is a glycolipid, phospholipid, oligosaccharide, G protein coupled cell receptor (GPCR), differentiation cluster (CD) cell surface protein, cell surface receptor The nucleic acid molecule according to claim 1 or 2, which specifically binds to one or more cell membrane components selected from the group consisting of a cell surface co-receptor and a protein. (A) the first sequence segment encoding the ER signal sequence comprises or consists of the nucleic acid sequence set forth in SEQ ID NO: 1;
(B) the third sequence segment encoding a linker comprises or consists of the nucleic acid sequence shown in SEQ ID NO: 3; and / or (c) the fourth sequence segment encoding the VSV-G Comprising or consisting of the nucleic acid sequence set forth in SEQ ID NO: 5,
The nucleic acid molecule according to any one of claims 1 to 3.   A vector comprising the nucleic acid molecule according to claim 1.   A host cell comprising the nucleic acid molecule according to any one of claims 1 to 4 or the vector according to claim 5.   A polypeptide encoded by the nucleic acid molecule of any of claims 1-4. A (poly) peptide comprising or consisting of a cell membrane binding domain encoded by the nucleic acid molecule according to any one of claims 1 to 4, or a VSV-G linked to the polypeptide according to claim 7. A method,
The method comprises culturing the host cell of claim 6 and isolating the manufactured VSV-G or manufactured polypeptide linked to a (poly) peptide comprising or consisting of a cell membrane binding domain. Method. (A) a VSV-G linked to a (poly) peptide comprising and / or consisting of a cell membrane-binding domain encoded by a nucleic acid molecule according to any of claims 1-4; and (b) a cell membrane-binding domain. VSV-G not linked to (poly) peptide comprising and / or consisting of
Pseudotyped lentiviral vector particles. A method for producing pseudotyped lentiviral vector particles according to claim 9, comprising:
The method comprises: (i) one or more packaging plasmids encoding a viral particle protein in the host cell;
(Ii) a vector comprising the nucleic acid molecule according to any of claims 1 to 4; and (iii) a nucleic acid molecule encoding VSV-G not linked to a (poly) peptide comprising or consisting of a cell membrane binding domain. A method comprising transfecting a vector comprising: A method for transducing cells comprising:
The method comprises the step of transducing a cell to be transduced by contacting the cell to be transduced with a pseudotyped lentiviral vector particle according to claim 9 under conditions suitable for transduction.   12. The method of claim 11, further comprising contacting the cell with an adjuvant, preferably a poloxamer having a molecular weight of 12.8 kDa to about 15 kDa.   13. The method of claim 12, further comprising spinoculating pseudotyped lentiviral vector particles with the cells prior to, simultaneously with, or after contacting the target cells with the adjuvant.   The cells to be transduced are selected from the group consisting of tumor cells, lymphoid cells, epithelial cells, neurons and stem cells, and / or preferably the cells to be transduced are part of a heterogeneous cell population The method according to claim 11, wherein (A) (i) a nucleic acid molecule according to any one of claims 1 to 4; or (a) (ii) a nucleic acid molecule according to any one of claims 1 to 4 and a cell membrane binding domain. A nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding VSV-G not linked to a (poly) peptide;
And / or (b) (i) the vector according to claim 5; or (b) (ii) the vector according to claim 5 and a (poly) peptide comprising or consisting of a cell membrane binding domain A vector comprising a nucleic acid molecule comprising or consisting of a nucleic acid sequence encoding VSV-G;
And / or (c) (i) a host cell according to claim 6; or (c) (ii) a host cell according to claim 6 and a (poly) peptide comprising or consisting of a cell membrane binding domain. A host cell comprising a vector comprising a nucleic acid molecule comprising or consisting of a non-VSV-G encoding nucleic acid sequence;
And / or (d) (i) a polypeptide according to claim 7; or (d) (ii) a polypeptide according to claim 7 and a (poly) peptide comprising or consisting of a cell membrane binding domain. Not VSV-G;
And / or (e) pseudotyped lentiviral vector particles according to claim 9;
In addition, if necessary, a kit containing instructions for use.